Method for producing an l-amino acid using bacterium of the enterobacteriaceae family with attenuated expression of a gene coding for small rna

ABSTRACT

The present invention provides a method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family, particularly a bacterium belonging to genus  Escherichia  or  Pantoea , which has been modified to attenuate expression of a gene coding for sRNA.

This application is a continuation under 35 U.S.C. §120 of U.S. patentapplication Ser. No. 12/212,743, filed on Sep. 18, 2008, which was acontinuation under 35 U.S.C. §120 of PCT Patent Application No.PCT/JP2007/056752, filed on Mar. 22, 2007, which claimed priority under35 U.S.C. §119(a) to Russian Patent Application No. 2006109062, filed onMar. 23, 2006, Russian Patent Application No. 2006109063, filed on Mar.23, 2006, Russian Patent Application No. 2006111808, filed on Apr. 11,2006, Russian Patent Application No. 2006111809, filed on Apr. 11, 2006,Russian Patent Application No. 2006115067, filed on May 4, 2006, RussianPatent Application No. 2006115068, filed on May 4, 2006, Russian PatentApplication No. 2006115070, filed on May 4, 2006, Russian PatentApplication No. 2006119216, filed on Jun. 2, 2006, Russian PatentApplication No. 2006123751, filed on Jul. 4, 2006, Russian PatentApplication No. 2007101437, filed on Jan. 16, 2007, and Russian PatentApplication No. 2007101440, filed on Jan. 16, 2007, the entireties ofwhich are hereby incorporated by reference. The Sequence Listing filedelectronically herewith is also hereby incorporated by reference in itsentirety (File Name: 2010-08-18T_US-275D_Seq_List; File Size: 30 KB;Date Created: Aug. 18, 2010).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the microbiological industry, andspecifically to a method for producing an L-amino acid using a bacteriumof the Enterobacteriaceae family which has been modified to attenuateexpression of a gene coding for small RNA (sRNA).

2. Brief Description of the Related Art

Conventionally, L-amino acids are industrially produced by fermentationmethods utilizing strains of microorganisms obtained from naturalsources, or mutants thereof. Typically, the microorganisms are modifiedto enhance production yields of L-amino acids.

Many techniques to enhance L-amino acid production yields have beenreported, including transformation of microorganisms with recombinantDNA (see, for example, U.S. Pat. No. 4,278,765). Other techniques forenhancing production yields include increasing the activities of enzymesinvolved in amino acid biosynthesis and/or desensitizing the targetenzymes of the feedback inhibition by the resulting L-amino acid (see,for example, WO 95/16042 or U.S. Pat. Nos. 4,346,170; 5,661,012 and6,040,160).

Another way to enhance L-amino acid production yields is to attenuateexpression of a gene or several genes which are involved in degradationof the target L-amino acid, genes which are responsible for divertingthe precursors of the target L-amino acid from the L-amino acidbiosynthetic pathway, genes which are involved in the redistribution ofcarbon, nitrogen, and phosphate fluxes, and genes coding for toxins etc.

Small, untranslated RNAs are present in many different organisms,ranging from bacteria to mammals. These RNAs carry out a variety ofbiological functions. Many of them can function as regulators of geneexpression at the posttranscriptional level, either by acting asantisense RNAs, by binding to complementary sequences of targettranscripts, or by interacting with proteins. Regulatory RNAs areinvolved in the control of a large variety of processes such as plasmidreplication, transposition in pro- and eukaryotes, phage development,viral replication, bacterial virulence, global circuits in bacteria inresponse to environmental changes, or developmental control in lowereukaryotes (Argaman L. et. al., Current Biology, 11: 941-50 (2001)).

Small RNA (sRNA) molecules have gained much interest recently. ManyEscherichia coli genes are known to code for sRNAs: c0067, c0293, c0299,c0343, c0362, c0465, c0614, c0664, c0719, csrB, dicF, dsrA, ffs, gadY,gcvB, is092, is102, is 128, isrA, micC, micF, oxyS, rnpB, rprA, rybA,rybB, rydB, ryeA, ryeB, ryeC, ryeD, ryeE, ryfA, rygB, rygC, rygD, sgrS,spf, sraA, sraB, sraD, sraE, sraG, sraH, sraI, sraJ, sraK, sraL, sroA,sroB, sroC, sroD, sroE, sroF, sroG, sroH, ssrA, ssrS, t44(tff), tp2,tpke11, tpke70 (Hershberg, R., et. al., Nucleic Acids Res.,31(7):1813-20 (2003) and Vogel, J., et al, Nucleic Acids Res., 31(22):6435-43 (2003)). Most of these genes are still uncharacterized and theircellular roles are unknown. Traditionally, most RNA molecules werethought to function as mediators that carry the information from thegene to the translational machinery. Exceptions were the transfer RNAsand ribosomal RNAs that had long been known to have functions of theirown, associated also with translation. However, it is now widelyacknowledged that other types of untranslated RNA molecules (sRNA) existthat are involved in a diverse range of functions, from structuralthrough regulatory to catalytic (Hershberg, R., et al., Nucleic AcidsRes. 31(7): 1813-1820 (2003)).

The sraE and rygB genes encode small, untranslated RNAs—SraE and RygB ofapproximately 89 nt and 83 nt in length, respectively, which are encodedwithin the same inter-ORF region of the genome. Interactions between theSraE RNA and Hfq protein and between the RygB RNA and Hfq have beendetected, SraE RNA and RygB RNA bound Hfq quite efficiently (>30% bound)(Wassarman, K. M. et al, Genes Dev. 1; 15(13):1637-51 (2001)). There issome sequence similarity between sraE and rygB, and they are transcribedin the same direction. SraE and rygB, which are located in the sameintergenic region between aas and galR, show significant sequencesimilarity of 77% identity over 84 nt (Hershbserg, R., et. al., NucleicAcids Res., 31(7):1813-20 (2003)). Despite this high sequencesimilarity, these two sRNAs exhibit an almost mutually exclusiveexpression pattern: RygB levels increase around the onset of thestationary phase and decrease thereafter (Vogel, J., et al, NucleicAcids Res., 31(22): 6435-43 (2003)), whereas SraE is produced asstationary phase progresses (Argaman, L. et al, Current Biology, 11:941-50 (2001)).

The sroE gene encodes a small, untranslated RNA called SroE. SroE sRNAwas shown to be processed from a longer transcript, that is, theupstream gcpE gene. Its 5′ end was mapped to the UAA stop codon of gcpE(third nucleotide). SroE extends into the promoter region downstream ofthe hisS gene. Both adjacent genes and the 110 bp IGR are conservedbetween E. coli and Salmonella species; the SroE sequences are predictedto fold into identical two-stem-loop structures with any sequencevariation confined to the loops. The rybB gene is considered to be agenuine sRNA gene. RybB is produced as a shorter processed RNA specieslate in growth. The estimated half life was determined in stationaryphase to be 8 min. The half life of SraE in stationary phase is 16minutes; the half life of RygB in stationary phase is 30 minutes. SraHis one of the most stable known sRNA. The half life of SraH instationary phase is 32 minutes (Vogel, J., et al, Nucleic Acids Res.,31(22): 6435-43 (2003)).

Expression of sraE is not affected by heat or cold shock treatmentduring early growth. The promoter of the sraE gene is found to be activein vitro, and the transcript length is similar to that observed in vivo.Expression of the E. coli K12 sraA gene was investigated in cells grownto different growth phases in either rich or minimal media supplementedwith glycerol and in cells subjected to heat shock or cold shocktreatment. The transcript levels of sraA were constant regardless of theconditions. SraB RNA is expressed during the stationary phase only andis at the highest levels at 8 and 10 hr after dilution of the culture.The gcvB gene is expressed in the early logarithmic phase, but itsproduction slows with cellular growth. It was found that most of thegcvB transcripts read through the first terminator and stop at thesecond one, and thus result in an RNA product of 205 nucleotides. GcvBRNA is not affected by heat or cold shock treatment during early growth.Minor increases in GcvB expression were detected during the stationaryphase when the cells were grown in glycerol minimal medium. SraH RNA ishighly abundant during the stationary phase, but low levels can bedetected in exponentially growing cells as well. Expression of sraH isnot affected by heat or cold shock treatment during early growth. Invitro transcription of sraH resulted in a product of approximately 120nucleotides, which corresponds to the predicted full-length RNA(Argaman, L. et al, Current Biology, 11: 941-50 (2001)). An interactionbetween RyhA (SraH) RNA and Hfq, a small, highly abundant RNA-bindingprotein, has been detected. High-copy expression of ryhA (sraH) causesincreased expression of rpoS in minimal media (Wassarman, K. M. et al,Genes Dev. 1; 15(13):1637-51 (2001)).

The dsrA gene encodes DsrA RNA, a small (87-nt) regulatory RNA of E.coli that acts via RNA-RNA interactions to control translation andturnover of specific mRNAs. Two targets of DsrA regulation are RpoS, thestationary-phase and stress response sigma factor (sigmas), and H-NS, ahistone-like nucleoid protein and global transcription repressor (LeaseR. A., et al, Proc. Natl. Acad. Sci. USA, 95(21):12456-61 (1998)). Genesregulated globally by RpoS and H-NS include stress response proteins andvirulence factors for pathogenic E. coli. Genes induced by DsrA havebeen identified by using transcription profiling via DNA arrays (LeaseR. A., et al, J. Bacteriol., 186(18):6179-85 (2004)). Steady-statelevels of mRNAs from many genes increased with DsrA overproduction,including multiple acid resistance genes of E. coli. Quantitative primerextension analysis verified the induction of individual acid resistancegenes in the hdeAB, gadAX, and gadBC operons. Overproduction of DsrAfrom a plasmid rendered the acid-sensitive dsrA mutant extremely acidresistant, confirming that DsrA RNA plays a regulatory role in acidresistance.

Both the rate of transcription initiation of the dsrA gene and thestability of DsrA RNA are regulated by temperature, increasing at lowtemperature (Repoila F. and Gottesman S., J. Bacteriol., 183(13):4012-23(2001)). The dsrA promoter is temperature-sensitive (Repoila F. andGottesman S., J. Bacteriol., 185(22):6609-14 (2003)).

DsrA RNA acts by base-pairing to activate or repress translation, or todestabilize mRNAs. Base-pairing between this regulatory RNA and itstarget mRNAs requires the Sm-like Hfq protein, which most likelyfunctions as an RNA chaperone to increase RNA unfolding or local targetRNA concentration (Storz G., et al, Curr. Opin. Microbiol., 7(2):140-44(2004)).

The rprA gene encodes a 106 nucleotide regulatory RNA called RprA. Aswith DsrA, RprA is predicted to form three stem-loop structures. Atleast two small RNAs, DsrA and RprA, participate in the positiveregulation of the stationary phase sigma factor RpoS translation. UnlikeDsrA, RprA does not have an extensive region of complementarity to theRpoS leader, leaving its mechanism of action unclear. It was assumedthat RprA is non-essential in the positive regulation (Majdalani, N., etal., Mol. Microbiol, 39(5), 1382-94 (2001)).

The E. coli gcvB gene encodes a small RNA transcript that is nottranslated in vivo. Transcription from the gcvB promoter is activated bythe GcvA protein and repressed by the GcvR protein, both of which arethe transcriptional regulators of the gcvTHP operon which encodes theenzymes of the glycine cleavage system. A strain carrying a chromosomaldeletion of gcvB exhibits normal regulation of gcvTHP expression andglycine cleavage enzyme activity. However, this mutant has highconstitutive synthesis of OppA and DppA, which are periplasmic-bindingprotein components of two major peptide transport systems which arenormally repressed in cells growing in rich medium. The alteredregulation of oppA and dppA was also demonstrated using oppA-phoA anddppA-lacZ gene fusions. Although the mechanism(s) involved in therepression by gcvB of these two genes is not known, oppA regulationappears to be at the translational level, whereas dppA regulation occursat the mRNA level. The sequence of gcvB was shown to contain two sitesfor transcription termination (M. L. Urbanowski et al, Mol. Microbiol.,37: 856-68 (2000)).

The micC gene (IS063) encodes a ˜100-nucleotide small-RNA transcript.The expression of this small RNA is increased at a low temperature andin minimal medium. Twenty-two nucleotides at the 5′ end of thistranscript have the potential to form base pairs with the leadersequence of the mRNA encoding the outer membrane protein OmpC. MicC wasshown to inhibit ribosome binding to the ompC mRNA leader in vitro andto require the Hfq RNA chaperone to function (Chen, S., et al., J.Bacteriol., 186(20):6679-80 (2004)).

The ryeE gene encodes a small, untranslated RNA-RyeE RNA 86 nt inlength. All known sRNA are encoded within intergenic (Ig) regions(defined as regions between ORFs). The Ig region corresponding to ryeEis highly conserved when compared to the closely related Salmonella andKlebsiella pneumonia species. An interaction between RyeE RNA and Hfqprotein has been detected, RyeE RNA bounds Hfq quite efficiently (>30%bound). Overproduction of RyeE causes decreased expression of rpoSduring the stationary phase in LB (Wassarman, K. M., Genes Dev., 15(13):1637-51 (2001)).

But currently, there have been no reports of inactivating a gene codingfor sRNA for the purpose of producing L-amino acids.

SUMMARY OF THE INVENTION

Aspects of the present invention include enhancing the productivity ofL-amino acid-producing strains and providing a method for producing anL-amino acid using these strains.

Attenuating expression of a gene coding for sRNA can enhance productionof L-amino acids, such as L-threonine, L-lysine, L-cysteine,L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, L-serine,L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid,L-proline, L-arginine, L-phenylalanine, L-tyrosine, and L-tryptophan.

The present invention provides a bacterium of the Enterobacteriaceaefamily having an increased ability to produce amino acids, such asL-threonine, L-lysine, L-cysteine, L-methionine, L-leucine,L-isoleucine, L-valine, L-histidine, L-serine, L-alanine, L-asparagine,L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, L-arginine,L-phenylalanine, L-tyrosine, and L-tryptophan.

It is an aspect of the present invention to provide an L-aminoacid-producing bacterium of the Enterobacteriaceae family, wherein thebacterium has been modified to attenuate expression of a gene coding forsRNA.

It is a further aspect of the present invention to provide the bacteriumas described above, wherein said gene coding for sRNA is selected fromthe group consisting of c0067, c0293, c0299, c0343, c0362, c0465, c0614,c0664, c0719, csrB, dicF, dsrA, ffs, gadY, gcvB, is092, is102, is128,isrA, micC, micF, oxyS, rnpB, rprA, rybA, rybB, rydB, ryeA, ryeB, ryeC,ryeD, ryeE, ryfA, rygB, rygC, rygD, sgrS, spf, sraA, sraB, sraD, sraE,sraG, sraH, sraI, sraJ, sraK, sraL, sroA, sroB, sroC, sroD, sroE, sroF,sroG, sroH, ssrA, ssrS, t44(tff), tp2, tpke11, and tpke70.

It is a further aspect of the present invention to provide the bacteriumas described above, wherein said expression of a gene coding for sRNA isattenuated by inactivating the gene coding for sRNA.

It is a further aspect of the present invention to provide the bacteriumas described above, wherein the bacterium belongs to the genusEscherichia.

It is a further aspect of the present invention to provide the bacteriumas described above, wherein the bacterium belongs to the genus Pantoea.

It is a further aspect of the present invention to provide the bacteriumas described above, wherein said L-amino acid is selected from the groupconsisting of an aromatic L-amino acid and a non-aromatic L-amino acid.

It is a further aspect of the present invention to provide the bacteriumas described above, wherein said aromatic L-amino acid is selected fromthe group consisting of L-phenylalanine, L-tyro sine, and L-tryptophan.

It is a further aspect of the present invention to provide the bacteriumas described above, wherein said non-aromatic L-amino acid is selectedfrom the group consisting of L-threonine, L-lysine, L-cysteine,L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, L-serine,L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid,L-proline, and L-arginine.

It is a further aspect of the present invention to provide a method forproducing an L-amino acid comprising:

cultivating the bacterium as described above in a medium, and

collecting said L-amino acid from the medium.

It is a further aspect of the present invention to provide the method asdescribed above, wherein said L-amino acid is selected from the groupconsisting of an aromatic L-amino acid and a non-aromatic L-amino acid.

It is a further aspect of the present invention to provide the method asdescribed above, wherein said aromatic L-amino acid is selected from thegroup consisting of L-phenylalanine, L-tyrosine, and L-tryptophan.

It is a further aspect of the present invention to provide the method asdescribed above, wherein said non-aromatic L-amino acid is selected fromthe group consisting of L-threonine, L-lysine, L-cysteine, L-methionine,L-leucine, L-isoleucine, L-valine, L-histidine, L-serine, L-alanine,L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline,and L-arginine.

The present invention is described in detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the relative positions of primers P1 (upstream primer) andP2 (downstream primer) on plasmid pMW118-attL-Cm-attR which is used as atemplate for PCR amplification of the cat gene.

FIG. 2 shows the construction of the chromosomal DNA fragment containingthe inactivated target gene.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 1. Bacterium of thePresent Invention

The bacterium of the present invention is an L-amino acid-producingbacterium of the Enterobacteriaceae family, wherein the bacterium hasbeen modified to attenuate expression of the a gene coding for sRNA(small RNA).

The phrase “L-amino acid-producing bacterium” means a bacterium whichhas an ability to produce and excrete an L-amino acid into a medium,when the bacterium is cultured in the medium.

The term “L-amino acid-producing bacterium” also means a bacterium whichis able to produce and cause accumulation of an L-amino acid in aculture medium in an amount larger than a wild-type or parental strainof E. coli, such as E. coli K-12, and preferably means that themicroorganism is able to cause accumulation in a medium of the targetL-amino acid in an amount not less than 0.5 g/L, more preferably notless than 1.0 g/L. The term “L-amino acid” includes L-alanine,L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid,L-glutamine, L-histidine, L-isoleucine, L-leucine, L-lysine,L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine,L-tryptophan, L-tyrosine, and L-valine.

The term “aromatic L-amino acid” includes L-phenylalanine, L-tyrosine,and L-tryptophan. The term “non-aromatic L-amino acid” includesL-threonine, L-lysine, L-cysteine, L-methionine, L-leucine,L-isoleucine, L-valine, L-histidine, L-serine, L-alanine, L-asparagine,L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, andL-arginine. L-threonine, L-lysine, L-cysteine, L-leucine, L-histidine,L-glutamic acid, L-phenylalanine, L-tryptophan, L-proline, andL-arginine are particularly preferred.

The Enterobacteriaceae family includes bacteria belonging to the generaEscherichia, Enterobacter, Erwinia, Klebsiella, Pantoea, Photorhabdus,Providencia, Salmonella, Serratia, Shigella, Morganella, Yersinia, etc.Specifically, those classified into the Enterobacteriaceae according tothe taxonomy used by the NCBI (National Center for BiotechnologyInformation) database(http://www.ncbi.nlm.nih.gov/htbinpost/Taxonomy/wgetorg?mode=Tree&id=1236&1v1=3&keep=1&srchmode=1&unlock)can be used. A bacterium belonging to the genus Escherichia or Pantoeais preferred.

The phrase “a bacterium belonging to the genus Escherichia” means thatthe bacterium is classified into the genus Escherichia according to theclassification known to a person skilled in the art of microbiology.Examples of a bacterium belonging to the genus Escherichia as used inthe present invention include, but are not limited to, Escherichia coli(E. coli).

The bacterium belonging to the genus Escherichia is not particularlylimited, however for example, bacteria described by Neidhardt, F. C. etal. (Escherichia coli and Salmonella typhimurium, American Society forMicrobiology, Washington D.C., 1208, Table 1) are encompassed by thepresent invention.

The phrase “a bacterium belonging to the genus Pantoea” means that thebacterium is classified as the genus Pantoea according to theclassification known to a person skilled in the art of microbiology.Some species of Enterobacter agglomerans have been recentlyre-classified into Pantoea agglomerans, Pantoea ananatis, Pantoeastewartii or the like, based on the nucleotide sequence analysis of 16SrRNA, etc. (Int. J. Syst. Bacteriol., 43, 162-173 (1993)).

The phrase “a gene coding for sRNA” means a gene encoding an RNA that isnot translated into a protein and has a small size, preferably 50 to 500bases in length.

The phrase “bacterium has been modified to attenuate expression of agene coding for sRNA” means that the bacterium has been modified in sucha way that the modified bacterium contains a reduced amount of the sRNA,as compared with an unmodified bacterium, or is unable to synthesize thesRNA.

The phrase “inactivation of a gene coding for sRNA” means that themodified DNA region is unable to naturally express the gene due to thedeletion of a part of the gene or of the gene entirely, or themodification of an adjacent region of the gene, including sequencescontrolling gene expression, such as promoters, enhancers, attenuators,etc.

The level of gene expression can be determined by measuring the amountof sRNA transcribed from the gene using various known methods includingNorthern blotting, quantitative RT-PCR, and the like.

The c0067 gene encodes the C0067 RNA. The c0067 gene (nucleotides inpositions 238,462 to 238,586; GenBank accession no. NC_(—)000913.2; gi:49175990) is located between the yafT ORF and the yafU ORF on thechromosome of E. coli K-12. The nucleotide sequence of the c0067 gene isshown in SEQ ID NO: 69.

The c0293 gene encodes the C0293 RNA. The c0293 gene (nucleotides inpositions 1,195,937 to 1,196,009; GenBank accession no. NC_(—)000913.2;gi: 49175990) is located between the icd gene and the ymfD ORF on thechromosome of E. coli K-12. The nucleotide sequence of the c0293 gene isshown in SEQ ID NO: 70.

The c0299 gene encodes the C0299 RNA. The c0299 gene (nucleotides inpositions 1,229,852 to 1,229,930; GenBank accession no. NC_(—)000913.2;gi: 49175990) is located between the hlyE gene and the umuD gene on thechromosome of E. coli K-12. The nucleotide sequence of the c0299 gene isshown in SEQ ID NO:71.

The c0343 gene encodes the C0343 RNA. The c0343 gene (nucleotides inpositions 1,407,387 to 1,407,461; GenBank accession no. NC_(—)000913.2;gi: 49175990) is located between the ydaN ORF and the dbpA gene on thechromosome of E. coli K-12. The nucleotide sequence of the c0343 gene isshown in SEQ ID NO:72.

The c0362 gene encodes the C0362 RNA. The c0362 gene (nucleotides inpositions 1,550,025 to 1,550,410; GenBank accession no. NC_(—)000913.2;gi: 49175990) is located between the fdnI gene and the yddM ORF on thechromosome of E. coli K-12. The nucleotide sequence of the c0362 gene isshown in SEQ ID NO: 1.

The c0465 gene encodes the C0465 RNA. The c0465 gene (nucleotides inpositions 1,970,763 to 1,970,840; GenBank accession no. NC_(—)000913.2;gi: 49175990) is located between the tar gene and the cheW gene on thechromosome of E. coli K-12. The nucleotide sequence of the c0465 gene isshown in SEQ ID NO: 2.

The c0614 gene encodes the C0614 RNA. The c0614 gene (nucleotidescomplemented to nucleotides in positions 2,651,474 to 2,651,560; GenBankaccession no. NC_(—)000913.2; gi: 49175990) is located between the sseAgene and the IS128 gene, overlapping with the IS128 gene, on thechromosome of E. coli K-12. The nucleotide sequence of the c0614 gene isshown in SEQ ID NO:73.

The c0664 gene encodes the C0664 RNA. The c0664 gene (nucleotides inpositions 2,833,077 to 2,833,189; GenBank accession no. NC_(—)000913.2;gi: 49175990) is located between the ygbD gene and the hypF gene on thechromosome of E. coli K-12. The nucleotide sequence of the c0664 gene isshown in SEQ ID NO: 74.

The c0719 gene encodes the C0719 RNA. The c0719 gene (nucleotides inpositions 3,119,380 to 3,119,601; GenBank accession no. NC_(—)000913.2;gi: 49175990) is located between the glcA gene and the glcB gene on thechromosome of E. coli K-12. The nucleotide sequence of the c0719 gene isshown in SEQ ID NO: 75.

The csrB gene (synonyms—ECK2787, b4408) encodes the CsrB RNA. The csrBgene (nucleotides complementary to nucleotides in positions 2,922,178 to2,922,537; GenBank accession no. NC_(—)000913.2; gi: 49175990) islocated between the yqcC ORF and the syd gene on the chromosome of E.coli K-12. The nucleotide sequence of the csrB gene is shown in SEQ IDNO: 76.

The dicF gene (synonyms—ECK1568, b1574) encodes the DicF RNA. The dicFgene (nucleotides in positions 1,647,406 to 1,647,458; GenBank accessionno. NC_(—)000913.2; gi: 49175990) is located between the rzpQ gene andthe dicB gene on the chromosome of E. coli K-12. The nucleotide sequenceof the dicF gene is shown in SEQ ID NO: 77.

The dsrA gene (synonym—b1954) encodes DsrA RNA, a global regulator ofgene expression. The dsrA gene (nucleotides in positions 2,023,336 to2,023,250; GenBank accession no. NC_(—)000913.2; gi:49175990) is locatedbetween the yodD and yedP genes on the E. coli strain K-12 chromosome.The nucleotide sequence of the dsrA gene is shown in SEQ ID NO: 3.

The ffs gene (synonyms—ECK0449, b0455) encodes the Ffs RNA. The ffs gene(nucleotides in positions 475,672 to 475,785; GenBank accession no.NC_(—)000913.2; gi: 49175990) is located between the ybaZ ORF and theybaA ORF on the chromosome of E. coli K-12. The nucleotide sequence ofthe ffs gene is shown in SEQ ID NO: 78.

The gadY gene (synonyms—ECK3500, b4452, IS183) encodes the GadY RNA. ThegadY gene (nucleotides in positions 1,647,406 to 1,647,458; GenBankaccession no. NC_(—)000913.2; gi: 49175990) is located between the rzpQgene and the dicB gene on the chromosome of E. coli K-12. The nucleotidesequence of the gadY gene is shown in SEQ ID NO: 79.

The gcvB gene (synonyms: ECK2804, psrA11, IS145, b4443) encodes the GcvBRNA. The gcvB gene (nucleotides in positions 2,940,718 to 2,940,922;GenBank accession no. NC_(—)000913.2; gi: 16130715) is located betweenthe gcvA gene and the ygdI ORF on the chromosome of E. coli K-12. Thenucleotide sequence of the gcvB gene is shown in SEQ ID NO: 4.

The IS092 gene (synonyms—ECK1902, b4434) encodes the IS092 RNA. TheIS092 gene (nucleotides complementary to nucleotides in positions1,985,863 to 1,986,022; GenBank accession no. NC_(—)000913.2; gi:49175990) is located between the yecJ ORF and the yecR ORF on thechromosome of E. coli K-12. The nucleotide sequence of the IS092 gene isshown in SEQ ID NO: 80.

The IS102 gene (synonyms—ECK1992, b4435) encodes the IS102 RNA. TheIS102 gene (nucleotides in positions 2,069,339 to 2,069,542; GenBankaccession no. NC_(—)000913.2; gi: 49175990) is located between the yeePORF and the flu gene on the chromosome of E. coli K-12. The nucleotidesequence of the IS102 gene is shown in SEQ ID NO: 81.

The IS128 gene encodes the IS128 RNA. The IS128 gene (nucleotides inpositions 2,651,537 to 2,651,745; GenBank accession no. NC_(—)000913.2;gi: 49175990) is located between the c0614 gene and the ryfA gene,overlapping with the c0614 gene, on the chromosome of E. coli K-12. Thenucleotide sequence of the IS128 gene is shown in SEQ ID NO: 82.

The isrA gene (synonyms—ECK1336, b4426, IS061) encodes the IsrA RNA. TheisrA gene (nucleotides complemented to nucleotides in positions1,403,676 to 1,403,833; GenBank accession no. NC_(—)000913.2; gi:49175990) is located between the abgR gene and the ydaL ORF on thechromosome of E. coli K-12. The nucleotide sequence of the isrAgene isshown in SEQ ID NO: 83.

The micC gene (synonyms: ECK1373, IS063, tke8, b4427) encodes the MicCRNA. The micC gene (nucleotides in positions 1,435,145 to 1,435,253;GenBank accession no. NC_(—)000913.2; gi: 16127999) is located betweenthe gene ompN and the ORF ydbK on the chromosome of E. coli K-12. Thenucleotide sequence of the micC gene is shown in SEQ ID NO: 5.

The micF gene (synonyms: ECK2208, stc, b4439) encodes the MicF RNA. ThemicF gene (nucleotides in positions 2,311,106 to 2,311,198; GenBankaccession no. NC_(—)000913.2; gi: 16127999) is located between the ompCgene and the rcsD gene on the chromosome of E. coli K-12. The nucleotidesequence of the micF gene is shown in SEQ ID NO: 84.

The oxyS gene (synonyms—ECK3952, b4458) encodes the OxyS RNA. The oxySgene (nucleotides complementary to nucleotides in positions 4,156,308 to4,156,417; GenBank accession no. NC_(—)000913.2; gi: 49175990) islocated between the argH gene and the oxyR gene on the chromosome of E.coli K-12. The nucleotide sequence of the oxyS gene is shown in SEQ IDNO: 85.

The rnpB gene (synonyms—ECK3111, b3123) encodes the RnpB RNA. The rnpBgene (nucleotides complementary to nucleotides in positions 3,268,238 to3,268,614; GenBank accession no. NC_(—)000913.2; gi: 49175990) islocated between the yhaC ORF and the garK gene on the chromosome of E.coli K-12. The nucleotide sequence of the rnpB gene is shown in SEQ IDNO: 86.

The rprA gene (synonyms—ECK1686, psrA5, IS083, b4431) encodes the RprARNA The rprA gene of E. coli (nucleotides in positions 1,768,395 to1,768,499; GenBank accession no. NC_(—)000913.2; gi:49175990) is locatedbetween the ydiK ORF and the ydiL ORF on the chromosome of E. coli K-12.The nucleotide sequence of the rprA gene is shown in SEQ ID NO:6.

The rybA gene (synonyms: ECK0806, b4416) encodes the RybA RNA. The rybAgene (nucleotides complementary to nucleotides in positions 852,175 to852,263; GenBank accession no. NC_(—)000913.2; gi:16127999) is locatedbetween the yliL ORF and the mntR gene on the chromosome of E. coliK-12. The nucleotide sequence of the rybA gene is shown in SEQ ID NO:87.

The rybB gene (synonyms: p25, b4417) encodes the RybB RNA. The rybB gene(nucleotides in positions 887,198 to 887,276; GenBank accession no.NC_(—)000913.2; gi:16127999) is located between the ORF ybjK and the ORFybjL on the chromosome of E. coli K-12. The nucleotide sequence of therybB gene is shown in SEQ ID NO: 7.

The rydB gene (synonyms: ECK1681, tpe7, IS082, b4430) encodes the RydBRNA. The rydB gene (nucleotides complemented to nucleotides in positions1,762,737 to 1,762,804; GenBank accession no. NC_(—)000913.2;gi:16127999) is located between the sufA gene and the ydiH ORF on thechromosome of E. coli K-12. The nucleotide sequence of the rydB gene isshown in SEQ ID NO: 88.

The ryeA gene (synonyms: ECK1838, sraC, sraCryeA, psrA8, tkpe79, IS091,b4432) encodes the RyeA RNA. The ryeA gene (nucleotides in positions1,921,090 to 1,921,338; GenBank accession no. NC_(—)000913.2;gi:16127999) is located between the pphA gene and the yebY ORF,interlapping the ryeB gene which is oriented in the oppositeorientation, on the chromosome of E. coli K-12. The nucleotide sequenceof the ryeA gene is shown in SEQ ID NO: 89.

The ryeB gene (synonyms: ECK1839, tkpe79, IS091, b4433) encodes the RyeBRNA. The ryeB gene (nucleotides complementary to nucleotides inpositions 1,921,188 to 1,921,308; GenBank accession no. NC_(—)000913.2;gi:16127999) is located in the region of the ryeA gene on the chromosomeof E. coli K-12. The nucleotide sequence of the ryeB gene is shown inSEQ ID NO: 90.

The ryeC gene (synonyms: ECK2068, QUAD1a, tp11, b4436) encodes the RyeCRNA. The ryeC gene (nucleotides in positions 2,151,299 to 2,151,447;GenBank accession no. NC_(—)000913.2; gi:16127999) is located betweenthe yegL ORF and the ryeD gene on the chromosome of E. coli K-12. Thenucleotide sequence of the ryeC gene is shown in SEQ ID NO: 91.

The ryeD gene (synonyms: ECK2069, QUAD1b, tpe60, b4437) encodes the RyeDRNA. The ryeD gene (nucleotides in positions 2,151,634 to 2,151,776;GenBank accession no. NC_(—)000913.2; gi:16127999) is located betweenthe ryeC gene and the mdtA gene on the chromosome of E. coli K-12. Thenucleotide sequence of the ryeDgene is shown in SEQ ID NO: 92.

The ryeE gene (synonyms: ECK2078, b4438) encodes the RyeE RNA. The ryeEgene (nucleotides in positions 2,165,136 to 2,165,221; GenBank accessionno. NC_(—)000913.2; gi: 49175990) is located between the yegQ ORF andthe ogrK gene on the chromosome of E. coli K-12. The nucleotide sequenceof the ryeE gene is shown in SEQ ID NO: 8.

The ryfA gene (synonyms: ECK2518, b4440) encodes the RyeE RNA. The ryeEgene (nucleotides in positions 2,651,877-2,652,180; GenBank accessionno. NC_(—)000913.2; gi: 49175990) is located between the yegQ ORF andthe ogrK gene on the chromosome of E. coli K-12. The nucleotide sequenceof the ryfA gene is shown in SEQ ID NO: 93.

The rygB gene (synonyms: ECK2834, PAIR2, t59, b4445, omrB) encodes theRygB RNA. The rygB gene (nucleotides complemented to nucleotides inpositions 2,974,332 to 2,974,407; GenBank accession no. NC_(—)000913.2;gi: 49175990) is located between the sraE gene and the galR gene on thechromosome of E. coli K-12. The nucleotide sequence of the rygB gene isshown in SEQ ID NO: 9. The rygB gene may be attenuated together with theadjacent sraE gene.

The rygC gene (synonyms: ECK2908, QUAD1c, t27, b4446) encodes the RygCRNA. The rygC gene (nucleotides in positions 3,054,837 to 3,054,987;GenBank accession no. NC_(—)000913.2; gi: 49175990) is located betweenthe sraE gene and the galR gene on the chromosome of E. coli K-12. Thenucleotide sequence of the rygC gene is shown in SEQ ID NO: 94.

The rygD gene (synonyms: ECK3041, tp8, C0730, IS156, QUAD1d, b4447)encodes the RygD RNA. The rygD gene (nucleotides complementary tonucleotides in positions 3,192,773 to 3,192,992; GenBank accession no.NC_(—)000913.2; gi: 49175990) is located between the sraE gene and thegalR gene on the chromosome of E. coli K-12. The nucleotide sequence ofthe rygD gene is shown in SEQ ID NO: 95.

The sgrS gene (synonyms: ECK0071, ryaA, b4577) encodes the SgrS RNA. ThesgrS gene (nucleotides in positions 77,367 to 77,593; GenBank accessionno. NC_(—)000913.2; gi: 49175990) is located between the sgrR gene andthe setA gene on the chromosome of E. coli K-12. The nucleotide sequenceof the sgrS gene is shown in SEQ ID NO: 96.

The spf gene (synonyms: ECK3856, b3864, spot42) encodes the Spf RNA. Thespf gene (nucleotides in positions 4,047,922 to 4,048,030; GenBankaccession no. NC_(—)000913.2; gi: 49175990) is located between the polAgene and the yihA ORF on the chromosome of E. coli K-12. The nucleotidesequence of the spf gene is shown in SEQ ID NO: 97.

The sraA gene (synonyms: psrA3, t15) encodes the SraA RNA. The sraA gene(nucleotides complementary to nucleotides in positions 457,952 to458,008; GenBank accession no. NC_(—)000913.2; gi: 49175990) is locatedbetween the clpX gene and the lon gene on the chromosome of E. coliK-12. The nucleotide sequence of the sraA gene is shown in SEQ ID NO:10.

The sraB gene (synonyms: psrA4, pke20) encodes the SraB RNA. The sraBgene (nucleotides in positions 1,145,811 to 1,145,979; GenBank accessionno. NC_(—)000913.2; gi:49175990) is located on the chromosome of E. coliK-12 upstream, but overlapping with the yceD ORF, which is oriented inthe opposite direction and located upstream of sraB. The nucleotidesequence of the sraB gene is shown in SEQ ID NO: 11.

The sraD gene (synonyms: micA, ECK2682, psrA10, b4442) encodes the SraDRNA. The sraD gene (nucleotides in positions 2,812,823 to 2,812,897;GenBank accession no. NC_(—)000913.2; gi: 49175990) is located betweenthe luxS gene and the gshA gene on the chromosome of E. coli K-12. Thenucleotide sequence of the sraD gene is shown in SEQ ID NO: 98.

The sraE gene (synonyms: ECK2833, psrA12, rygA, PAIR2, t59, b4444, omrA)encodes the SraE RNA. The sraE gene (nucleotides complemented tonucleotides in positions 2,974,124 to 2,974,211; GenBank accession no.NC_(—)000913.2; gi: 49175990) is located between the aas gene and therygB gene on the chromosome of E. coli K-12. The nucleotide sequence ofthe rygB gene is shown in SEQ ID NO: 12. The sraE gene may be attenuatedtogether with the adjacent rygB gene.

The sraG gene (synonyms: ECK3153, psrA15, p3, b4449) encodes the SraGRNA. The sraG gene (nucleotides in positions 3,309,247 to 3,309,420;GenBank accession no. NC_(—)000913.2; gi: 49175990) is located betweenthe pnp gene and the rpsO gene, overlapping with the pnp gene, on thechromosome of E. coli K-12. The nucleotide sequence of the sraG gene isshown in SEQ ID NO: 99.

The sraH gene (synonyms: ECK3199, psrA16, ryhA, b4450) encodes the SraHRNA. The sraH gene (nucleotides in positions 3,348,599 to 3,348,706;GenBank accession no. NC_(—)000913.2; gi: 49175990) is located betweenthe elbB gene and the arcB gene on the chromosome of E. coli K-12. Thenucleotide sequence of the sraH gene is shown in SEQ ID NO: 13.

The sraI gene (synonyms: ECK3426, psrA18, IS176, b4451, ryhB) encodesthe SraI RNA. The sraI gene (nucleotides complemented to nucleotides inpositions 3,578,946 to 3,579,039; GenBank accession no. NC_(—)000913.2;gi: 49175990) is located between the yhhX ORF and the yhhY ORF on thechromosome of E. coli K-12. The nucleotide sequence of the sraI gene isshown in SEQ ID NO: 100.

The sraJ gene (synonyms: ECK3795, psrA20, ryiA, k19, b4456) encodes theSraJ RNA. The sraJ gene (nucleotides in positions 3,984,455 to3,984,626; GenBank accession no. NC_(—)000913.2; gi: 49175990) islocated between the aslA gene and the hemY gene on the chromosome of E.coli K-12. The nucleotide sequence of the sraJ gene is shown in SEQ IDNO: 101.

The sraK gene (synonyms: ECK3858, psrA21, ryiB, tpk2, IS198, b4457,csrC) encodes the SraK RNA. The sraK gene (nucleotides in positions4,049,059 to 4,049,303; GenBank accession no. NC_(—)000913.2; gi:49175990) is located between the yihA ORF and the yihi ORF on thechromosome of E. coli K-12. The nucleotide sequence of the sraK gene isshown in SEQ ID NO: 102.

The sraL gene (synonyms: ECK4056, psrA24, ryjA, b4459) encodes the SraLRNA. The sraL gene (nucleotides complemented to nucleotides in positions4,275,950 to 4,276,089; GenBank accession no. NC_(—)000913.2; gi:49175990) is located between the soxR gene and the yjcD ORF, overlappingwith the soxR gene, on the chromosome of E. coli K-12. The nucleotidesequence of the sraL gene is shown in SEQ ID NO: 103.

The sroA gene (synonym tpe79) encodes the SroA RNA. The sroA gene(nucleotides complementary to nucleotides in positions 75,516 to 75,608;GenBank accession no. NC_(—)000913.2; gi: 49175990) is located betweenthe tbpA gene and the sgrR gene on the chromosome of E. coli K-12. Thenucleotide sequence of the sroA gene is shown in SEQ ID NO: 104.

The sroB gene encodes the SroB RNA. The sroB gene (nucleotides inpositions 506,428 to 506,511; GenBank accession no. NC_(—)000913.2; gi:49175990) is located between the ybaK ORF and the ybaP ORF, overlappingwith the ybaP ORF, on the chromosome of E. coli K-12. The nucleotidesequence of the sroB gene is shown in SEQ ID NO: 105.

The sroC gene (synonym HB_(—)314) encodes the SroC RNA. The sroC gene(nucleotides complemented to nucleotides in positions 685,904 to686,066; GenBank accession no. NC_(—)000913.2; gi: 49175990) is locatedbetween the gltJ gene and the gltI gene, overlapping with the gltI gene,on the chromosome of E. coli K-12. The nucleotide sequence of the sroCgene is shown in SEQ ID NO: 106.

The sroD gene (synonym p24) encodes the SroD RNA. The sroD gene(nucleotides complemented to nucleotides in positions 1,886,041 to1,886,126; GenBank accession no. NC_(—)000913.2; gi: 49175990) islocated between the rnd gene and the fadD gene, overlapping with thefadD gene, on the chromosome of E. coli K-12. The nucleotide sequence ofthe sroD gene is shown in SEQ ID NO: 107.

The sroE gene (synonym k20) encodes the SroE RNA (synonym k20). The sroEgene (nucleotides complementary to nucleotides in positions 2,638,617 to2,638,708; GenBank accession no. NC_(—)000913.2; gi: 49175990) islocated between the hisS gene and the ispG gene on the chromosome of E.coli K-12. The nucleotide sequence of the sroE gene is shown in SEQ IDNO: 14.

The sroF gene (synonyms: ECK2554, b4441, tke1) encodes the SroF RNA. ThesroF gene (nucleotides complementary to nucleotides in positions 2689362to 2689214); GenBank accession no. NC_(—)000913.2; gi: 49175990) islocated between the yfhK ORF and the purL gene on the chromosome of E.coli K-12. The nucleotide sequence of the sroF gene is shown in SEQ IDNO: 108.

The sroG gene (synonym HB_(—)456) encodes the SroG RNA. The sroG gene(nucleotides complemented to nucleotides in positions 3,182,592 to3,182,740; GenBank accession no. NC_(—)000913.2; gi: 49175990) islocated between the ribB gene and the yqiC ORF on the chromosome of E.coli K-12. The nucleotide sequence of the sroG gene is shown in SEQ IDNO: 109.

The sroH gene encodes the SroH RNA. The sroH gene (nucleotidescomplemented to nucleotides in positions 4,188,350 to 4,188,510; GenBankaccession no. NC_(—)000913.2; gi: 49175990) is located between the htrCgene and the thiH gene, overlapping with the htrC gene, on thechromosome of E. coli K-12. The nucleotide sequence of the sroH gene isshown in SEQ ID NO: 110.

The ssrA gene (synonyms: ECK2617, b262, sipB) encodes the SsrA RNA. ThessrA gene (nucleotides in positions 2,753,615 to 2,753,977; GenBankaccession no. NC_(—)000913.2; gi: 49175990) is located between the smpBand intA genes on the chromosome of E. coli K-12. The nucleotidesequence of the ssrA gene is shown in SEQ ID NO: 111.

The ssrS gene (synonyms: ECK2906, b2911, ssr) encodes the SsrS RNA. ThessrS gene (nucleotides in positions 3,054,005 to 3,054,187; GenBankaccession no. NC_(—)000913.2; gi: 49175990) is located between the zapAgene and the ygfA ORF on the chromosome of E. coli K-12. The nucleotidesequence of the ssrS gene is shown in SEQ ID NO: 112.

The tff gene (synonyms: ECK0167, b4414, T44) encodes the tff RNA. Thetff gene (nucleotides in positions 189,712 to 189,847; GenBank accessionno. NC_(—)000913.2; gi: 49175990) is located between the map gene andthe rsp gene on the chromosome of E. coli K-12. The nucleotide sequenceof the tff gene is shown in SEQ ID NO: 113.

The tp2 gene encodes the Tp2 RNA. The tp2 gene (nucleotides complementedto nucleotides in positions 122,697 to 122,857; GenBank accession no.NC_(—)000913.2; gi: 49175990) is located between the pdhR gene and theaceE gene, overlapping with the pdhR gene, on the chromosome of E. coliK-12. The nucleotide sequence of the tp2 gene is shown in SEQ ID NO:114.

The tpke11 gene encodes the Tpke11 RNA. The tpke11 gene (nucleotidescomplemented to nucleotides in positions 14,080 to 14,168; GenBankaccession no. NC_(—)000913.2; gi: 49175990) is located between the dnaKgene and the dnaJ gene on the chromosome of E. coli K-12. The nucleotidesequence of the tpke11 gene is shown in SEQ ID NO: 115.

The tpke70 gene encodes the Tp70 RNA. The tpke70 gene (nucleotidescomplementary to nucleotides in positions 2,494,216 to 2,494,651;GenBank accession no. NC_(—)000913.2; gi: 49175990) is located betweenthe lpxP gene and the yfdZ ORF, overlapping with the lpxP gene, on thechromosome of E. coli K-12. The nucleotide sequence of the tpke70 geneis shown in SEQ ID NO: 116.

Since there may be some differences in DNA sequences between the generaor strains of the Enterobacteriaceae family, the gene coding for sRNA isnot limited to the genes shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14 and SEQ ID NO: 69-116, but may include geneshomologous to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14 and SEQ ID NO: 69-116. Such a homologous gene may have homology thatis enough to recombine a sRNA gene on the chromosome of a hostbacterium.

Therefore, a gene coding for sRNA may be a variant which hybridizesunder stringent conditions with a complement of the nucleotide sequenceshown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ IDNO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 andSEQ ID NO: 69-116, or a probe which can be prepared from the nucleotidesequence, provided that it encodes a functional sRNA prior toinactivation, or provided that attenuation of expression of the gene ina host bacterium leads to improvement of L-amino acid-producing abilityof the host bacterium. “Stringent conditions” include those under whicha specific hybrid, for example, a hybrid having homology of not lessthan 80%, preferably not less than 90%, more preferably not less than95%, still more preferably not less than 97%, and most preferably notless than 98%, is formed and a non-specific hybrid, for example, ahybrid having homology lower than the above, is not formed. For example,stringent conditions are exemplified by washing one time or more,preferably two or three times at a salt concentration of 1×SSC, 0.1%SDS, preferably 0.1×SSC, 0.1% SDS at 60° C. Duration of washing dependson the type of membrane used for blotting and, as a rule, should be whatis recommended by the manufacturer. For example, the recommendedduration of washing for the Hybond™ N+ nylon membrane (Amersham) understringent conditions is 15 minutes. Preferably, washing may be performed2 to 3 times. The length of the probe may be suitably selected,depending on the hybridization conditions, in this specific case it maybe about 100 bp.

Homology between two amino acid sequences can be determined using thewell-known methods, for example, the computer program BLAST 2.0.

Expression of a gene coding for sRNA can be attenuated by introducing amutation into the gene on the chromosome so that the intracellularamount of the sRNA encoded by the gene is decreased as compared to anunmodified strain. Such a mutation can be introduction of insertion of adrug-resistance gene, or deletion of a part of the gene or the entiregene (Qiu, Z. and Goodman, M. F., J. Biol. Chem., 272, 8611-8617 (1997);Kwon, D. H. et al, J. Antimicrob. Chemother., 46, 793-796 (2000)).Nucleotide sequence information of the sRNA gene and its flankingregions can be obtained based on the accession number of each sRNA genein Genbank. Expression of a gene coding for sRNA can also be attenuatedby modifying an expression regulating sequence such as the promoter, theShine-Dalgarno (SD) sequence, etc. (WO95/34672, Carrier, T. A. andKeasling, J. D., Biotechnol Prog 15, 58-64 (1999)).

For example, the following methods may be employed to introduce amutation by gene recombination. A mutant gene is prepared, and abacterium is transformed with a DNA fragment containing the mutant gene.Then, the native gene on the chromosome is replaced with the mutant geneby homologous recombination, and the resulting strain is selected. Suchgene replacement by homologous recombination can be conducted byemploying a linear DNA, which is known as “Red-driven integration”(Datsenko, K. A. and Wanner, B. L., Proc. Natl. Acad. Sci. USA, 97, 12,p 6640-6645 (2000)), or by methods employing a plasmid containing atemperature-sensitive replication (U.S. Pat. No. 6,303,383 or JP05-007491A). Furthermore, the incorporation of a site-specific mutationby gene substitution using homologous recombination such as set forthabove can also be conducted with a plasmid lacking the ability toreplicate in the host.

Expression of a gene can also be attenuated by insertion of a transposonor an IS factor into the coding region of the gene (U.S. Pat. No.5,175,107), or by conventional methods, such as mutagenesis treatmentusing UV irradiation or nitrosoguanidine(N-methyl-N′-nitro-N-nitrosoguanidine) treatment.

The presence or absence of a gene coding for sRNA in the chromosome of abacterium can be detected by well-known methods, including PCR, Southernblotting and the like. In addition, the level of gene expression can beestimated by measuring the amount of the RNA transcribed from the geneusing various well-known methods, including Northern blotting,quantitative RT-PCR, and the like.

Methods for preparation of plasmid DNA, digestion and ligation of DNA,transformation, selection of an oligonucleotide as a primer, and thelike may be ordinary methods well-known to one skilled in the art. Thesemethods are described, for instance, in Sambrook, J., Fritsch, E. F.,and Maniatis, T., “Molecular Cloning: A Laboratory Manual, SecondEdition”, Cold Spring Harbor Laboratory Press (1989).

L-Amino Acid-Producing Bacteria

As a bacterium of the present invention which is modified to attenuateexpression of a gene coding for sRNA, bacteria which are able to produceeither an aromatic or a non-aromatic L-amino acids may be used. Examplesof L-amino acid-producing bacteria are shown below.

The bacterium of the present invention can be obtained by attenuatingexpression of a gene coding for sRNA in a bacterium which inherently hasthe ability to produce L-amino acids. Alternatively, the bacterium ofpresent invention can be obtained by imparting the ability to produceL-amino acids to a bacterium already having the attenuated expression ofa gene coding for sRNA.

L-Threonine-Producing Bacteria

Examples of parent strains for deriving the L-threonine-producingbacteria of the present invention include, but are not limited to,strains belonging to the genus Escherichia, such as E. coli TDH-6/pVIC40(VKPM B-3996) (U.S. Pat. No. 5,175,107, U.S. Pat. No. 5,705,371), E.coli 472T23/pYN7 (ATCC 98081) (U.S. Pat. No. 5,631,157), E. coliNRRL-21593 (U.S. Pat. No. 5,939,307), E. coli FERM BP-3756 (U.S. Pat.No. 5,474,918), E. coli FERM BP-3519 and FERM BP-3520 (U.S. Pat. No.5,376,538), E. coli MG442 (Gusyatiner et al., Genetika (in Russian), 14,947-956 (1978)), E. coli VL643 and VL2055 (EP 1149911 A), and the like.

The strain TDH-6 is deficient in the thrC gene, as well as beingsucrose-assimilative, and the ilvA gene has a leaky mutation. Thisstrain also has a mutation in the rhtA gene, which imparts resistance tohigh concentrations of threonine or homoserine. The strain B-3996contains the plasmid pVIC40 which was obtained by inserting a thrA*BCoperon which includes a mutant thrA gene into a RSF1010-derived vector.This mutant thrA gene encodes aspartokinase homoserine dehydrogenase Iwhich has substantially desensitized feedback inhibition by threonine.The strain B-3996 was deposited on Nov. 19, 1987 in the All-UnionScientific Center of Antibiotics (Russia, 117105 Moscow, NagatinskayaStreet 3-A) under the accession number RIA 1867. The strain was alsodeposited in the Russian National Collection of IndustrialMicroorganisms (VKPM) (Russia, 117545 Moscow, 1^(st) Dorozhny proezd, 1)on Apr. 7, 1987 under the accession number VKPM B-3996.

E. coli VKPM B-5318 (EP 0593792B) may also be used as a parent strainfor deriving L-threonine-producing bacteria of the present invention.The strain B-5318 is prototrophic with regard to isoleucine, and atemperature-sensitive lambda-phage C1 repressor and PR promoter replacesthe regulatory region of the threonine operon in plasmid pVIC40. Thestrain VKPM B-5318 was deposited in the Russian National Collection ofIndustrial Microorganisms (VKPM) on May 3, 1990 under accession numberof VKPM B-5318.

Preferably, the bacterium of the present invention is additionallymodified to enhance expression of one or more of the following genes:

the mutant thrA gene which codes for aspartokinase homoserinedehydrogenase I resistant to feed back inhibition by threonine;

the thrB gene which codes for homoserine kinase;

the thrC gene which codes for threonine synthase;

the rhtA gene which codes for a putative transmembrane protein;

the asd gene which codes for aspartate-β-semialdehyde dehydrogenase; and

the aspC gene which codes for aspartate aminotransferase (aspartatetransaminase);

The thrA gene which encodes aspartokinase homoserine dehydrogenase I ofEscherichia coli has been elucidated (nucleotide positions 337 to 2799,GenBank accession NC_(—)000913.2, gi: 49175990). The thrA gene islocated between the thrL and thrB genes on the chromosome of E. coliK-12. The thrB gene which encodes homoserine kinase of Escherichia colihas been elucidated (nucleotide positions 2801 to 3733, GenBankaccession NC_(—)000913.2, gi: 49175990). The thrB gene is locatedbetween the thrA and thrC genes on the chromosome of E. coli K-12. ThethrC gene which encodes threonine synthase of Escherichia coli has beenelucidated (nucleotide positions 3734 to 5020, GenBank accessionNC_(—)000913.2, gi: 49175990). The thrC gene is located between the thrBgene and the yaaX open reading frame on the chromosome of E. coli K-12.All three genes functions as a single threonine operon. To enhanceexpression of the threonine operon, the attenuator region which affectsthe transcription is desirably removed from the operon (WO2005/049808,WO2003/097839).

A mutant thrA gene which codes for aspartokinase homoserinedehydrogenase I resistant to feed back inhibition by threonine, as wellas, the thrB and thrC genes can be obtained as one operon fromwell-known plasmid pVIC40 which is presented in the threonine producingE. coli strain VKPM B-3996. Plasmid pVIC40 is described in detail inU.S. Pat. No. 5,705,371.

The rhtA gene exists at 18 min on the E. coli chromosome close to theglnHPQ operon, which encodes components of the glutamine transportsystem. The rhtA gene is identical to ORF1 (ybiF gene, nucleotidepositions 764 to 1651, GenBank accession number AAA218541, gi:440181)and located between the pexB and ompX genes. The unit expressing aprotein encoded by the ORF1 has been designated the rhtA gene (rht:resistance to homoserine and threonine). Also, it was revealed that therhtA23 mutation is an A-for-G substitution at position −1 with respectto the ATG start codon (ABSTRACTS of the 17^(th) International Congressof Biochemistry and Molecular Biology in conjugation with Annual Meetingof the American Society for Biochemistry and Molecular Biology, SanFrancisco, Calif. Aug. 24-29, 1997, abstract No. 457, EP 1013765 A).

The asd gene of E. coli has already been elucidated (nucleotidepositions 3572511 to 3571408, GenBank accession NC_(—)000913.1,gi:16131307), and can be obtained by PCR (polymerase chain reaction;refer to White, T. J. et al., Trends Genet., 5, 185 (1989)) utilizingprimers prepared based on the nucleotide sequence of the gene. The asdgenes of other microorganisms can be obtained in a similar manner.

Also, the aspC gene of E. coli has already been elucidated (nucleotidepositions 983742 to 984932, GenBank accession NC_(—)000913.1,gi:16128895), and can be obtained by PCR. The aspC genes of othermicroorganisms can be obtained in a similar manner.

L-Lysine-Producing Bacteria

Examples of L-lysine-producing bacteria belonging to the genusEscherichia include mutants having resistance to an L-lysine analogue.The L-lysine analogue inhibits growth of bacteria belonging to the genusEscherichia, but this inhibition is fully or partially desensitized whenL-lysine coexists in a medium. Examples of the L-lysine analogueinclude, but are not limited to, oxalysine, lysine hydroxamate,S-(2-aminoethyl)-L-cysteine (AEC), γ-methyllysine, α-chlorocaprolactamand so forth. Mutants having resistance to these lysine analogues can beobtained by subjecting bacteria belonging to the genus Escherichia to aconventional artificial mutagenesis treatment. Specific examples ofbacterial strains useful for producing L-lysine include Escherichia coliAJ11442 (FERM BP-1543, NRRL B-12185; see U.S. Pat. No. 4,346,170) andEscherichia coli VL611. In these microorganisms, feedback inhibition ofaspartokinase by L-lysine is desensitized.

The strain WC196 may be used as an L-lysine producing bacterium ofEscherichia coli. This bacterial strain was bred by conferring AECresistance to the strain W3110, which was derived from Escherichia coliK-12. The resulting strain was designated Escherichia coli AJ13069strain and was deposited at the National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology (currentlyNational Institute of Advanced Industrial Science and Technology,International Patent Organism Depositary, Tsukuba Central 6, 1-1,Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on Dec. 6,1994 and received an accession number of FERM P-14690. Then, it wasconverted to an international deposit under the provisions of theBudapest Treaty on Sep. 29, 1995, and received an accession number ofFERM BP-5252 (U.S. Pat. No. 5,827,698).

Examples of parent strains for deriving L-lysine-producing bacteria ofthe present invention also include strains in which expression of one ormore genes encoding an L-lysine biosynthetic enzyme are enhanced.Examples of such genes include, but are not limited to, genes encodingdihydrodipicolinate synthase (dapA), aspartokinase (lysC),dihydrodipicolinate reductase (dapB), diaminopimelate decarboxylase(lysA), diaminopimelate dehydrogenase (ddh) (U.S. Pat. No. 6,040,160),phosphoenolpyrvate carboxylase (ppc), aspartate semialdehydedehydrogenease (asd), and aspartase (aspA) (EP 1253195 A). In addition,the parent strains may have an increased level of expression of the geneinvolved in energy efficiency (cyo) (EP 1170376 A), the gene encodingnicotinamide nucleotide transhydrogenase (pntAB) (U.S. Pat. No.5,830,716), the ybjE gene (WO2005/073390), or combinations thereof.

Examples of parent strains for deriving L-lysine-producing bacteria ofthe present invention also include strains having decreased oreliminated activity of an enzyme that catalyzes a reaction forgenerating a compound other than L-lysine by branching off from thebiosynthetic pathway of L-lysine. Examples of the enzymes that catalyzea reaction for generating a compound other than L-lysine by branchingoff from the biosynthetic pathway of L-lysine include homoserinedehydrogenase (WO 95/23864), lysine decarboxylase (U.S. Pat. No.5,827,698), and the malic enzyme (WO2005/010175).

L-Cysteine-Producing Bacteria

Examples of parent strains for deriving L-cysteine-producing bacteria ofthe present invention include, but are not limited to, strains belongingto the genus Escherichia, such as E. coli JM15 which is transformed withdifferent cysE alleles coding for feedback-resistant serineacetyltransferases (U.S. Pat. No. 6,218,168, Russian patent application2003121601); E. coli W3110 having over-expressed genes which encodeproteins suitable for secreting substances toxic for cells (U.S. Pat.No. 5,972,663); E. coli strains having lowered cysteine desulfohydraseactivity (JP11155571A2); E. coli W3110 with increased activity of apositive transcriptional regulator for cysteine regulon encoded by thecysB gene (W00127307 A1), and the like.

L-Leucine-Producing Bacteria

Examples of parent strains for deriving L-leucine-producing bacteria ofthe present invention include, but are not limited to, strains belongingto the genus Escherichia, such as E. coli strains resistant to leucine(for example, the strain 57 (VKPM B-7386, U.S. Pat. No. 6,124,121)) orleucine analogs including β-2-thienylalanine, 3-hydroxyleucine,4-azaleucine, 5,5,5-trifluoroleucine (JP 62-34397 B and JP 8-70879 A);E. coli strains obtained by the gene engineering method described inWO96/06926; E. coli H-9068 (JP 8-70879 A), and the like.

The bacterium of the present invention may be improved by enhancing theexpression of one or more genes involved in L-leucine biosynthesis.Examples include genes of the leuABCD operon, which are preferablyrepresented by a mutant leuA gene coding for isopropylmalate synthasefreed from feedback inhibition by L-leucine (U.S. Pat. No. 6,403,342).In addition, the bacterium of the present invention may be improved byenhancing the expression of one or more genes coding for proteins whichexcrete L-amino acid from the bacterial cell. Examples of such genesinclude the b2682 and b2683 genes (ygaZH genes) (EP 1239041 A2).

L-Histidine-Producing Bacteria

Examples of parent strains for deriving L-histidine-producing bacteriaof the present invention include, but are not limited to, strainsbelonging to the genus Escherichia, such as E. coli strain 24 (VKPMB-5945, RU2003677); E. coli strain 80 (VKPM B-7270, RU2119536); E. coliNRRL B-12116-B12121 (U.S. Pat. No. 4,388,405); E. coli H-9342 (FERMBP-6675) and H-9343 (FERM BP-6676) (U.S. Pat. No. 6,344,347); E. coliH-9341 (FERM BP-6674) (EP1085087); E. coli AI80/pFM201 (U.S. Pat. No.6,258,554) and the like.

Examples of parent strains for deriving L-histidine-producing bacteriaof the present invention also include strains in which expression of oneor more genes encoding an L-histidine biosynthetic enzyme are enhanced.Examples of such genes include genes encoding ATPphosphoribosyltransferase (hisG), phosphoribosyl AMP cyclohydrolase(hisI), phosphoribosyl-ATP pyrophosphohydrolase (hisIE),phosphoribosylformimino-5-aminoimidazole carboxamide ribotide isomerase(hisA), amidotransferase (hisH), histidinol phosphate aminotransferase(hisC), histidinol phosphatase (hisB), histidinol dehydrogenase (hisD),and so forth.

It is known that the L-histidine biosynthetic enzymes encoded by hisGand hisBHAFI are inhibited by L-histidine, and therefore anL-histidine-producing ability can also be efficiently enhanced byintroducing a mutation conferring resistance to the feedback inhibitioninto ATP phosphoribosyltransferase (Russian Patent Nos. 2003677 and2119536).

Specific examples of strains having an L-histidine-producing abilityinclude E. coli FERM-P 5038 and 5048 which have been introduced with avector carrying a DNA encoding an L-histidine-biosynthetic enzyme (JP56-005099 A), E. coli strains introduced with rht, a gene for an aminoacid-export (EP1016710A), E. coli 80 strain imparted withsulfaguanidine, DL-1,2,4-triazole-3-alanine, and streptomycin-resistance(VKPM B-7270, Russian Patent No. 2119536), and so forth.

L-Glutamic Acid-Producing Bacteria

Examples of parent strains for deriving L-glutamic acid-producingbacteria of the present invention include, but are not limited to,strains belonging to the genus Escherichia, such as E. coli VL334thrC⁺(EP 1172433). E. coli VL334 (VKPM B-1641) is an L-isoleucine andL-threonine auxotrophic strain having mutations in thrC and ilvA genes(U.S. Pat. No. 4,278,765). A wild-type allele of the thrC gene wastransferred by the method of general transduction using a bacteriophageP1 grown on the wild-type E. coli strain K12 (VKPM B-7) cells. As aresult, an L-isoleucine auxotrophic strain VL334thrC⁺ (VKPM B-8961),which is able to produce L-glutamic acid, was obtained.

Examples of parent strains for deriving the L-glutamic acid-producingbacteria of the present invention include, but are not limited to,strains in which expression of one or more genes encoding an L-glutamicacid biosynthetic enzyme are enhanced. Examples of such genes includegenes encoding glutamate dehydrogenase (gdhA), glutamine synthetase(glnA), glutamate synthetase (gltAB), isocitrate dehydrogenase (icdA),aconitate hydratase (acnA, acnB), citrate synthase (gltA),phosphoenolpyruvate carboxylase (ppc), pyruvate carboxylase (pyc),pyruvate dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA, pykF),phosphoenolpyruvate synthase (ppsA), enolase (eno), phosphoglyceromutase(pgmA, pgmI), phosphoglycerate kinase (pgk), glyceraldehyde-3-phophatedehydrogenase (gapA), triose phosphate isomerase (tpiA), fructosebisphosphate aldolase (fbp), phosphofructokinase (pfkA, pfkB), andglucose phosphate isomerase (pgi).

Examples of strains modified so that expression of the citratesynthetase gene, the phosphoenolpyruvate carboxylase gene, and/or theglutamate dehydrogenase gene is/are enhanced include those disclosed inEP1078989A, EP955368A, and EP952221A.

Examples of parent strains for deriving the L-glutamic acid-producingbacteria of the present invention also include strains having decreasedor eliminated activity of an enzyme that catalyzes synthesis of acompound other than L-glutamic acid by branching off from an L-glutamicacid biosynthesis pathway. Examples of such enzymes include isocitratelyase (aceA), α-ketoglutarate dehydrogenase (sucA),phosphotransacetylase (pta), acetate kinase (ack), acetohydroxy acidsynthase (ilvG), acetolactate synthase (ilvI), formate acetyltransferase(pfl), lactate dehydrogenase (ldh), and glutamate decarboxylase (gadAB).Bacteria belonging to the genus Escherichia deficient in theα-ketoglutarate dehydrogenase activity or having a reducedα-ketoglutarate dehydrogenase activity and methods for obtaining themare described in U.S. Pat. Nos. 5,378,616 and 5,573,945. Specifically,these strains include the following:

E. coli W3110sucA::Km^(R)

E. coli AJ12624 (FERM BP-3853)

E. coli AJ12628 (FERM BP-3854)

E. coli AJ12949 (FERM BP-4881)

E. coli W3110sucA::Km^(R) is a strain obtained by disrupting theα-ketoglutarate dehydrogenase gene (hereinafter referred to as “sucAgene”) of E. coli W3110. This strain is completely deficient inα-ketoglutarate dehydrogenase.

Other examples of L-glutamic acid-producing bacterium include thosewhich belong to the genus Escherichia and have resistance to an asparticacid antimetabolite. These strains can also be deficient in theα-ketoglutarate dehydrogenase activity and include, for example, E. coliAJ13199 (FERM BP-5807) (U.S. Pat. No. 5,908,768), FFRM P-12379, whichadditionally has a low L-glutamic acid decomposing ability (U.S. Pat.No. 5,393,671); AJ13138 (FERM BP-5565) (U.S. Pat. No. 6,110,714), andthe like.

Examples of L-glutamic acid-producing bacteria include mutant strainsbelonging to the genus Pantoea which are deficient in α-ketoglutaratedehydrogenase activity or have a decreased α-ketoglutarate dehydrogenaseactivity, and can be obtained as described above. Such strains includePantoea ananatis AJ13356. (U.S. Pat. No. 6,331,419). Pantoea ananatisAJ13356 was deposited at the National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology, Ministryof International Trade and Industry (currently, National Institute ofAdvanced Industrial Science and Technology, International PatentOrganism Depositary, Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi,Ibaraki-ken, 305-8566, Japan) on Feb. 19, 1998 under an accession numberof FERM P-16645. It was then converted to an international deposit underthe provisions of Budapest Treaty on Jan. 11, 1999 and received anaccession number of FERM BP-6615. Pantoea ananatis AJ13356 is deficientin the α-ketoglutarate dehydrogenase activity as a result of disruptionof the αKGDH-E1 subunit gene (sucA). The above strain was identified asEnterobacter agglomerans when it was isolated and deposited as theEnterobacter agglomerans AJ13356. However, it was recently re-classifiedas Pantoea ananatis on the basis of nucleotide sequencing of 16S rRNAand so forth. Although AJ13356 was deposited at the aforementioneddepository as Enterobacter agglomerans, for the purposes of thisspecification, they are described as Pantoea ananatis.

L-Phenylalanine-Producing Bacteria

Examples of parent strains for deriving L-phenylalanine-producingbacteria of the present invention include, but are not limited to,strains belonging to the genus Escherichia, such as E. coli AJ12739(tyrA::Tn10, tyrR) (VKPM B-8197); E. coli HW1089 (ATCC 55371) harboringthe mutant pheA34 gene (U.S. Pat. No. 5,354,672); E. coli MWEC101-b(KR8903681); E. coli NRRL B-12141, NRRL B-12145, NRRL B-12146 and NRRLB-12147 (U.S. Pat. No. 4,407,952). Also, as a parent strain, E. coliK-12 [W3110 (tyrA)/pPHAB (FERM BP-3566), E. coli K-12 [W3110(tyrA)/pPHAD] (FERM BP-12659), E. coli K-12 [W3110 (tyrA)/pPHATerm](FERM BP-12662) and E. coli K-12 [W3110 (tyrA)/pBR-aroG4, pACMAB] namedas AJ 12604 (FERM BP-3579) may be used (EP 488424 B1). Furthermore,L-phenylalanine producing bacteria belonging to the genus Escherichiawith an enhanced activity of the protein encoded by the yedA gene or theyddG gene may also be used (U.S. patent applications 2003/0148473 A1 and2003/0157667 A1).

L-Tryptophan-Producing Bacteria

Examples of parent strains for deriving the L-tryptophan-producingbacteria of the present invention include, but are not limited to,strains belonging to the genus Escherichia, such as E. coliJP4735/pMU3028 (DSM10122) and JP6015/pMU91 (DSM10123) deficient in thetryptophanyl-tRNA synthetase encoded by mutant trpS gene (U.S. Pat. No.5,756,345); E. coli SV164 (pGH5) having a serA allele encodingphosphoglycerate dehydrogenase free from feedback inhibition by serineand a trpE allele encoding anthranilate synthase free from feedbackinhibition by tryptophan (U.S. Pat. No. 6,180,373); E. coli AGX17(pGX44) (NRRL B-12263) and AGX6(pGX50)aroP (NRRL B-12264) deficient inthe enzyme tryptophanase (U.S. Pat. No. 4,371,614); E. coli AGX17/pGX50,pACKG4-pps in which a phosphoenolpyruvate-producing ability is enhanced(WO9708333, U.S. Pat. No. 6,319,696), and the like may be used.L-tryptophan-producing bacteria belonging to the genus Escherichia withan enhanced activity of the identified protein encoded by the yedA geneor the yddG gene may also be used (U.S. patent applications 2003/0148473A1 and 2003/0157667 A1).

Examples of parent strains for deriving the L-tryptophan-producingbacteria of the present invention also include strains in which one ormore activities of the enzymes selected from anthranilate synthase,phosphoglycerate dehydrogenase, and tryptophan synthase are enhanced.The anthranilate synthase and phosphoglycerate dehydrogenase are bothsubject to feedback inhibition by L-tryptophan and L-serine, so that amutation desensitizing the feedback inhibition may be introduced intothese enzymes. Specific examples of strains having such a mutationinclude an E. coli SV164 which harbors desensitized anthranilatesynthase and a transformant strain obtained by introducing into the E.coli SV164 the plasmid pGH5 (WO 94/08031), which contains a mutant serAgene encoding feedback-desensitized phosphoglycerate dehydrogenase.

Examples of parent strains for deriving the L-tryptophan-producingbacteria of the present invention also include strains into which thetryptophan operon which contains a gene encoding desensitizedanthranilate synthase has been introduced (JP 57-71397 A, JP 62-244382A, U.S. Pat. No. 4,371,614). Moreover, L-tryptophan-producing abilitymay be imparted by enhancing expression of a gene which encodestryptophan synthase, among tryptophan operons (trpBA). The tryptophansynthase consists of α and β subunits which are encoded by the trpA andtrpB genes, respectively. In addition, L-tryptophan-producing abilitymay be improved by enhancing expression of the isocitrate lyase-malatesynthase operon (WO2005/103275).

L-Proline-Producing Bacteria

Examples of parent strains for deriving L-proline-producing bacteria ofthe present invention include, but are not limited to, strains belongingto the genus Escherichia, such as E. coli 702ilvA (VKPM B-8012) which isdeficient in the ilvA gene and is able to produce L-proline (EP1172433). The bacterium of the present invention may be improved byenhancing the expression of one or more genes involved in L-prolinebiosynthesis. Examples of such genes for L-proline producing bacteriawhich are preferred include the proB gene coding for glutamate kinase ofwhich feedback inhibition by L-proline is desensitized (DE Patent3127361). In addition, the bacterium of the present invention may beimproved by enhancing the expression of one or more genes coding forproteins excreting L-amino acid from bacterial cell. Such genes areexemplified by b2682 and b2683 genes (ygaZH genes) (EP1239041 A2).

Examples of bacteria belonging to the genus Escherichia, which have anactivity to produce L-proline include the following E. coli strains:NRRL B-12403 and NRRL B-12404 (GB Patent 2075056), VKPM B-8012 (Russianpatent application 2000124295), plasmid mutants described in DE Patent3127361, plasmid mutants described by Bloom F. R. et al (The 15^(th)Miami winter symposium, 1983, p. 34), and the like.

L-Arginine-Producing Bacteria

Examples of parent strains for deriving L-arginine-producing bacteria ofthe present invention include, but are not limited to, strains belongingto the genus Escherichia, such as E. coli strain 237 (VKPM B-7925) (U.S.Patent Application 2002/058315 A1) and its derivative strains harboringmutant N-acetylglutamate synthase (Russian Patent Application No.2001112869), E. coli strain 382 (VKPM B-7926) (EP1170358A1), anarginine-producing strain into which argA gene encodingN-acetylglutamate synthetase is introduced therein (EP1170361A1), andthe like.

Examples of parent strains for deriving L-arginine producing bacteria ofthe present invention also include strains in which expression of one ormore genes encoding an L-arginine biosynthetic enzyme are enhanced.Examples of such genes include genes encoding N-acetylglutamyl phosphatereductase (argC), ornithine acetyl transferase (argJ), N-acetylglutamatekinase (argB), acetylornithine transaminase (argD), ornithine carbamoyltransferase (argF), argininosuccinic acid synthetase (argG),argininosuccinic acid lyase (argH), and carbamoyl phosphate synthetase(carAB).

L-Valine-Producing Bacteria

Examples of parent strains for deriving L-valine-producing bacteria ofthe present invention include, but are not limited to, strains whichhave been modified to overexpress the ilvGMEDA operon (U.S. Pat. No.5,998,178). It is desirable to remove the region of the ilvGMEDA operonwhich is required for attenuation so that expression of the operon isnot attenuated by L-valine that is produced. Furthermore, the ilvA genein the operon is desirably disrupted so that threonine deaminaseactivity is decreased.

Examples of parent strains for deriving L-valine-producing bacteria ofthe present invention include also include mutants having a mutation ofamino-acyl t-RNA synthetase (U.S. Pat. No. 5,658,766). For example, E.coli VL1970, which has a mutation in the ileS gene encoding isoleucinetRNA synthetase, can be used. E. coli VL1970 has been deposited in theRussian National Collection of Industrial Microorganisms (VKPM) (Russia,117545 Moscow, 1^(st) Dorozhny Proezd, 1) on Jun. 24, 1988 underaccession number VKPM B-4411.

Furthermore, mutants requiring lipoic acid for growth and/or lackingH⁺-ATPase can also be used as parent strains (WO96/06926).

L-Isoleucine-Producing Bacteria

Examples of parent strains for deriving L-isoleucine producing bacteriaof the present invention include, but are not limited to, mutants havingresistance to 6-dimethylaminopurine (JP 5-304969 A), mutants havingresistance to an isoleucine analogue such as thiaisoleucine andisoleucine hydroxamate, and mutants additionally having resistance toDL-ethionine and/or arginine hydroxamate (JP 5-130882 A). In addition,recombinant strains transformed with genes encoding proteins involved inL-isoleucine biosynthesis, such as threonine deaminase andacetohydroxate synthase, can also be used as parent strains (JP 2-458 A,FR 0356739, and U.S. Pat. No. 5,998,178).

2. Method of the Present Invention

The method of the present invention is a method for producing an L-aminoacid comprising cultivating the bacterium of the present invention in aculture medium to produce and excrete the L-amino acid into the medium,and collecting the L-amino acid from the medium.

The cultivation, collection, and purification of an L-amino acid fromthe medium and the like may be performed in a manner similar toconventional fermentation methods wherein an amino acid is producedusing a bacterium.

The medium used for culture may be either synthetic or natural, so longas it includes a carbon source and a nitrogen source and minerals and,if necessary, appropriate amounts of nutrients which the bacteriumrequires for growth. The carbon source may include various carbohydratessuch as glucose and sucrose, and various organic acids. Depending on themode of assimilation of the chosen microorganism, alcohol, includingethanol and glycerol, may be used. As the nitrogen source, variousammonium salts such as ammonia and ammonium sulfate, other nitrogencompounds such as amines, a natural nitrogen source such as peptone,soybean-hydrolysate, and digested fermentative microorganism can beused. As minerals, potassium monophosphate, magnesium sulfate, sodiumchloride, ferrous sulfate, manganese sulfate, calcium chloride, and thelike can be used. As vitamins, thiamine, yeast extract, and the like,can be used.

The cultivation is preferably performed under aerobic conditions, suchas a shaking culture, and a stirring culture with aeration, at atemperature of 20 to 40° C., preferably 30 to 38° C. The pH of theculture is usually between 5 and 9, preferably between 6.5 and 7.2. ThepH of the culture can be adjusted with ammonia, calcium carbonate,various acids, various bases, and buffers. Usually, a 1 to 5-daycultivation leads to accumulation of the target L-amino acid in theliquid medium.

After cultivation, solids such as cells can be removed from the liquidmedium by centrifugation or membrane filtration, and then the L-aminoacid can be collected and purified by ion-exchange, concentration,and/or crystallization methods.

EXAMPLES

The present invention will be more concretely explained below withreference to the following non-limiting Examples.

Example 1 Construction of a Strain with an Inactivated Gene Coding forsRNA 1. Deletion of a Target Gene

A strain having deletion of a gene coding for sRNA can be constructed bythe method initially developed by Datsenko, K. A. and Wanner, B. L.(Proc. Natl. Acad. Sci. USA, 2000, 97 (12), 6640-6645) called“Red-driven integration”. The DNA fragment containing the Cm^(R) markerencoded by the cat gene can be obtained by PCR, using primers P1(upstream primer) and P2 (downstream primer) and plasmidpMW118-attL-Cm-attR (WO 05/010175) as a template. Primer P1 containsboth a region complementary to the 35/36-nt region located at the 3′ endof a target gene and a region complementary to the attL region in theplasmid pMW118-attL-Cm-attR. Primer P2 contains both a regioncomplementary to the 35/36-nt region located at the 5′ end of a targetgene and a region complementary to the attR region in the plasmidpMW118-attL-Cm-attR. The nucleotide sequences of the regioncomplementary to the attL region in the plasmid pMW118-attL-Cm-attR andthe region complementary to the attR region in the plasmidpMW118-attL-Cm-attR are shown in SEQ ID NO: 15 and SEQ ID NO: 16,respectively.

Conditions for PCR can be as follows: denaturation step for 3 min at 95°C.; profile for two first cycles: 1 min at 95° C., 30 sec at 50° C., 40sec at 72° C.; profile for the last 25 cycles: 30 sec at 95° C., 30 secat 54° C., 40 sec at 72° C.; final step: 5 min at 72° C.

A PCR product (FIG. 1) can be obtained and purified in an agarose geland can be used for electroporation of the E. coli strain MG1655 (ATCC700926), which contains the plasmid pKD46 having a temperature-sensitivereplication. The plasmid pKD46 (Datsenko, K. A. and Wanner, B. L., Proc.Natl. Acad. Sci. USA, 2000, 97:12:6640-45) includes a 2,154 nucleotideDNA fragment of phage λ (nucleotide positions 31088 to 33241, GenBankaccession no. J02459), and contains genes of the X Red homologousrecombination system (γ, β, exo genes) under the control of thearabinose-inducible P_(araB) promoter. The plasmid pKD46 is necessaryfor integration of the PCR product into the chromosome of strain MG1655.

Electrocompetent cells can be prepared as follows: E. coli MG1655/pKD46can be grown overnight at 30° C. in LB medium containing ampicillin (100mg/l), and the culture can be diluted 100 times with 5 ml of SOB medium(Sambrook et al, “Molecular Cloning: A Laboratory Manual, SecondEdition”, Cold Spring Harbor Laboratory Press, 1989) containingampicillin and L-arabinose (1 mM). The cells can be grown with aerationat 30° C. to an OD₆₀₀ of ≈0.6 and then can be made electrocompetent byconcentrating 100-fold and washing three times with ice-cold deionizedH₂O. Electroporation can be performed using 70 μl of cells and ≈100 ngof the PCR product. Cells after electroporation can be incubated with 1ml of SOC medium (Sambrook et al, “Molecular Cloning: A LaboratoryManual, Second Edition”, Cold Spring Harbor Laboratory Press, 1989) at37° C. for 2.5 hours and then can be plated onto L-agar containingchloramphenicol (30 μg/ml) and grown at 37° C. to select Cm^(R)recombinants. Then, to eliminate the pKD46 plasmid, two passages onL-agar with Cm at 42° C. can be performed and the colonies can be testedfor sensitivity to ampicillin.

2. Verification of a Target Gene Deletion by PCR

The mutants having a gene coding for sRNA deleted and marked with the Cmresistance gene can be verified by PCR. Locus-specific primers P3 and P4can be used in PCR for the verification. Conditions for PCR verificationcan be as follows: denaturation step for 3 min at 94° C.; profile for 30cycles: 30 sec at 94° C., 30 sec at 54° C., 1 min at 72° C.; final step:7 min at 72° C. The PCR product obtained in the reaction with the cellsof parental strain MG1655 as a template and the PCR product obtained inthe reaction with the cells of mutant strain as the template shoulddiffer in length (FIG. 2). Mutant E. coli MG1655 having a gene codingfor sRNA (target gene) deleted and marked with the Cm resistance genecan be obtained in this manner.

3. Construction of Strains with Inactivated c0362, c0465, dsrA, gcvB,micC, rprA, rybB, ryeE, rygB-sraE, sraA, sraB, sraH and sroE genes

Strains having deletion of c0362, c0465, dsrA, gcvB, micC, rprA, rybB,ryeE, rygB-sraE, sraA, sraB, sraH and sroE genes were constructed by thedescribed above method. The nucleotide sequences of primers P1 (upstreamprimer) and P2 (downstream primer) used for PCR amplification of the catgene are shown in Table 1. The mutants having the target gene deletedand marked with the Cm resistance gene, constructed as a result ofelectroporation of E. coli strain MG1655 with 1699-bp PCR product,elimination the pKD46 plasmid and selection of mutants, were verified byPCR. Locus-specific primers P3 and P4 used in PCR for the verificationas well as lengths of PCR products obtained in the reactions with thecells of parental strain MG1655 and mutant strains as templates areshown in Table 1.

The mutant strains having deletion of c0362, c0465, dsrA, gcvB, micC,rprA, rybB, ryeE, rygB-sraE, sraA, sraB, sraH and sroE genes were namedMG1655 Δc0362::cat, MG1655 Δc0465::cat, MG1655 ΔdsrA::cat, MG1655ΔgcvB::cat, MG1655 ΔmicC::cat, MG1655 ΔrprA::cat, MG1655 ΔrybB::cat,MG1655 ΔryeE::cat, MG1655 ΔrygB-sraE::cat, MG1655 ΔsraA::cat, MG1655ΔsraB::cat, MG1655 ΔsraH::cat and MG1655 ΔsroE::cat, respectively.

TABLE 1 Length of PCR product obtained SEQ ID of upstream (P1) SEQ ID ofP3 and P4 using as a template and downstream (P2) primers forverification chromosome of: primers for of the target gene parentalmutant Target gene the target gene deletion deletion strain strain c0362P1-SEQ ID NO: 17 P3-SEQ ID NO: 19 ~0.7 kb ~2.0 kb P2-SEQ ID NO: 18P4-SEQ ID NO: 20 c0465 P1-SEQ ID NO: 21 P3-SEQ ID NO: 23 ~0.3 kb ~1.9 kbP2-SEQ ID NO: 22 P4-SEQ ID NO: 24 dsrA P1-SEQ ID NO: 25 P3-SEQ ID NO: 27~0.4 kbp ~2.0 kbp P2-SEQ ID NO: 26 P4-SEQ ID NO: 28 gcvB P1-SEQ ID NO:29 P3-SEQ ID NO: 31 ~0.5 kbp ~2.0 kbp P2-SEQ ID NO: 30 P4-SEQ ID NO: 32micC P1-SEQ ID NO: 33 P3-SEQ ID NO: 35 ~0.3 kbp ~1.9 kbp P2-SEQ ID NO:34 P4-SEQ ID NO: 36 rprA P1-SEQ ID NO: 37 P3-SEQ ID NO: 39 ~0.4 kbp ~1.9kbp P2-SEQ ID NO: 38 P4-SEQ ID NO: 40 rybB P1-SEQ ID NO: 41 P3-SEQ IDNO: 43 ~0.5 kbp ~2.1 kbp P2-SEQ ID NO: 42 P4-SEQ ID NO: 44 ryeE P1-SEQID NO: 45 P3-SEQ ID NO: 47 ~0.5 kbp ~2.2 kbp P2-SEQ ID NO: 46 P4-SEQ IDNO: 48 rygB- P1-SEQ ID NO: 49 P3-SEQ ID NO: 51 ~0.5 kbp ~1.9 kbp sraEP2-SEQ ID NO: 50 P4-SEQ ID NO: 52 sraA P1-SEQ ID NO: 53 P3-SEQ ID NO: 55~0.3 kbp ~1.9 kbp P2-SEQ ID NO: 54 P4-SEQ ID NO: 56 sraB P4-SEQ ID NO:57 P3-SEQ ID NO: 59 ~0.4 kbp ~2.0 kbp P2-SEQ ID NO: 58 P4-SEQ ID NO: 60sraH P1-SEQ ID NO: 61 P3-SEQ ID NO: 63 ~0.5 kbp ~2.1 kbp P2-SEQ ID NO:62 P4-SEQ ID NO: 64 sroE P1-SEQ ID NO: 65 P3-SEQ ID NO: 67 ~0.3 kbp ~1.9kbp P2-SEQ ID NO: 66 P4-SEQ ID NO: 68

Example 2 Production of L-Threonine by E. Coli Strain Having a GeneCoding for sRNA Deleted

To test the effect of inactivation of a gene coding for sRNA onthreonine production, DNA fragments from the chromosome of theabove-described mutant E. coli MG1655 having a gene coding for sRNA(target gene) deleted and marked with the Cm resistance gene can betransferred to the threonine-producing E. coli strain VKPM B-3996 by P1transduction (Miller, J. H. Experiments in Molecular Genetics, ColdSpring Harbor Lab. Press, 1972, Plainview, N.Y.) to obtain strainB-3996-Δtarget gene. The strain B-3996 was deposited on Nov. 19, 1987 inthe All-Union Scientific Center of Antibiotics (Russia, 117105 Moscow,Nagatinskaya Street, 3-A) under the accession number RIA 1867. Thestrain was also deposited in the Russian National Collection ofIndustrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1^(st) Dorozhnyproezd, 1) on Apr. 7, 1987 under the accession number B-3996. Thestrains B-3996-Δc0362, B-3996-Δc0465, B-3996-ΔdsrA, B-3996-ΔgcvB,B-3996-ΔmicC, B-3996-ΔrprA, B-3996-ΔrybB, B-3996-ΔryeE,B-3996-ΔrygB-sraE, B-3996-ΔsraB and B-3996-ΔsraH were obtained in thismanner.

Strains B-3996 and each of strains B-3996-Δc0362, B-3996-Δc0465,B-3996-ΔdsrA, B-3996-ΔgcvB, B-3996-ΔmicC, B-3996-ΔrprA, B-3996-ΔrybB,B-3996-ΔryeE, B-3996-ΔrygB-sraE, B-3996-ΔsraB or B-3996-ΔsraH, wereseparately grown for 18-24 hours at 37° C. on L-agar plates. To obtain aseed culture, the strains were grown on a rotary shaker (250 rpm) at 32°C. for 18 hours in 20×200-mm test tubes containing 2 ml of L-brothsupplemented with 4% glucose. Then, the fermentation medium wasinoculated with 0.21 ml (10%) of seed material. The fermentation wasperformed in 2 ml of minimal medium for fermentation in 20×200-mm testtubes. Cells were grown for 65 hours at 32° C. with shaking at 250 rpm.

After cultivation, the amount of L-threonine which had accumulated inthe medium, was determined by paper chromatography using the followingmobile phase: butanol-acetic acid-water=4:1:1 (v/v). A solution ofninhydrin (2%) in acetone was used as a visualizing reagent. A spotcontaining L-threonine was cut out, L-threonine was eluted with 0.5%water solution of CdCl₂, and the amount of L-threonine was estimatedspectrophotometrically at 540 nm. The results of ten (for B-3996-ΔdsrA,3996-ΔrprA and B-3996-ΔsraB)/eight (for B-3996-Δc0362, B-3996-Δc0465,B-3996-ΔgcvB, B-3996-ΔryeE, B-3996-ΔrygB-sraE and B-3996-ΔsraH)/five(for B-3996-ΔmicC and B-3996-ΔrybB) independent test tube fermentationsare shown in Table 2. As follows from Table 2, B-3996-Δc0362,B-3996-Δc0465, B-3996-ΔdsrA, B-3996-ΔgcvB, B-3996-ΔmicC, B-3996-ΔrprA,B-3996-ΔrybB, B-3996-ΔryeE, B-3996-ΔrygB-sraE, B-3996-ΔsraB andB-3996-ΔsraH produced a higher amount of L-threonine, as compared toB-3996.

TABLE 2 Amount of Strain OD₅₄₀ L-threonine, g/l B-3996 24.6 ± 0.6 24.7 ±0.3 B-3996-Δc0362 24.7 ± 0.7 26.8 ± 1.6 B-3996 29.5 ± 0.5 27.3 ± 0.2B-3996-Δc0465 29.4 ± 0.7 27.6 ± 0.6 B-3996 25.0 ± 1.1 29.0 ± 0.4B-3996-ΔgcvB 25.0 ± 0.5 31.0 ± 0.7 B-3996 28.3 ± 0.3 27.6 ± 0.7B-3996-ΔsraB 29.7 ± 0.6 28.5 ± 0.7 B-3996 32.8 ± 0.9 21.1 ± 0.3B-3996-ΔdsrA 32.2 ± 0.5 21.9 ± 0.6 B-3996 22.9 ± 1.5 25.5 ± 0.5B-3996-ΔrybB 18.5 ± 2.0 26.7 ± 0.5 B-3996 29.4 ± 0.7 25.9 ± 0.7B-3996-ΔrygB-sraE 30.3 ± 1.2 28.0 ± 1.1 B-3996-ΔryeE 28.5 ± 1.0 28.0 ±1.0 B-3996-ΔsraH 29.9 ± 1.2 27.1 ± 0.9 B-3996 24.4 ± 0.4 26.8 ± 0.5B-3996-ΔrprA 24.5 ± 1.2 27.6 ± 0.9 B-3996 19.3 ± 0.8 20.2 ± 0.9B-3996-ΔmicC 19.8 ± 0.9 24.3 ± 1.4

The composition of the fermentation medium (g/l) was as follows:

Glucose 80.0 (NH₄)₂SO₄ 22.0 NaCl 0.8 KH₂PO₄ 2.0 MgSO₄7H₂O 0.8 FeSO₄7H₂O0.02 MnSO₄5H₂O 0.02 Thiamine HCl 0.0002 Yeast extract 1.0 CaCO₃ 30.0

Glucose and magnesium sulfate were sterilized separately. CaCO₃ wassterilized by dry-heat at 180° C. for 2 hours. The pH was adjusted to7.0. The antibiotic was introduced into the medium after sterilization.

Example 3 Production of L-Lysine by E. coli Strain Having a Gene Codingfor sRNA Deleted

To test the effect of inactivation of a gene coding for sRNA on lysineproduction, DNA fragments from the chromosome of the above-describedmutant E. coli MG1655 having a gene coding for sRNA (target gene)deleted and marked with the Cm resistance gene can be transferred to thelysine-producing E. coli strain AJ11442 by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press,1972, Plainview, N.Y.) to obtain strain AJ11442-Δtarget gene. AJ11442strain was deposited at the National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology (currentlyNational Institute of Advanced Industrial Science and Technology,International Patent Organism Depositary, Tsukuba Central 6, 1-1,Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on May 1,1981 and received an accession number of FERM BP-1543. Both E. colistrains, AJ11442 and AJ11442-Δtarget gene, can be cultured in L-mediumat 37° C., and 0.3 ml of the obtained culture can be inoculated into 20ml of the fermentation medium containing the required drugs in a 500-mlflask. The cultivation can be carried out at 37° C. for 16 h by using areciprocal shaker at the agitation speed of 115 rpm. After thecultivation, the amounts of L-lysine and residual glucose in the mediumcan be measured by a known method (Biotech-analyzer AS210 manufacturedby Sakura Seiki Co.). Then, the yield of L-lysine can be calculatedrelative to consumed glucose for each of the strains.

The composition of the fermentation medium (g/l) is as follows:

Glucose 40 (NH₄)₂SO₄ 24 K₂HPO₄ 1.0 MgSO₄7H₂O 1.0 FeSO₄7H₂O 0.01MnSO₄5H₂O 0.01 Yeast extract 2.0

The pH is adjusted to 7.0 by KOH and the medium is autoclaved at 115° C.for 10 min. Glucose and MgSO₄7H₂O are sterilized separately. CaCO₃ isdry-heat sterilized at 180° C. for 2 hours and added to the medium for afinal concentration of 30 g/l.

Example 4 Production of L-Cysteine by E. coli Strain Having a GeneCoding for sRNA Deleted

To test the effect of inactivation of a gene coding for sRNA onL-cysteine production, DNA fragments from the chromosome of theabove-described mutant E. coli MG1655 having a gene coding for sRNA(target gene) deleted and marked with the Cm resistance gene can betransferred to the E. coli L-cysteine-producing strain JM15(ydeD) by P1transduction (Miller, J. H. Experiments in Molecular Genetics, ColdSpring Harbor Lab. Press, 1972, Plainview, N.Y.) to obtain the strainJM15(ydeD)-Δtarget gene.

E. coli strain JM15(ydeD) is a derivative of E. coli strain JM15 (U.S.Pat. No. 6,218,168) which can be transformed with DNA having the ydeDgene, which codes for a membrane protein, and is not involved in abiosynthetic pathway of any L-amino acid (U.S. Pat. No. 5,972,663,US2005-0124049). The strain JM15 (CGSC# 5042) can be obtained from TheColi Genetic Stock Collection at the E. coli Genetic Resource Center,MCD Biology Department, Yale University (http://cgsc.biology.yale.edu/).

Fermentation conditions for evaluation of L-cysteine production weredescribed in detail in Example 6 of U.S. Pat. No. 6,218,168.

Example 5 Production of L-Leucine by E. Coli Strain Having a Gene Codingfor sRNA Deleted

To test the effect of inactivation of a gene coding for sRNA onL-leucine production, DNA fragments from the chromosome of theabove-described mutant E. coli MG1655 having a gene coding for sRNA(target gene) deleted and marked with the Cm resistance gene can betransferred to the E. coli L-leucine-producing strain 57 (VKPM B-7386,U.S. Pat. No. 6,124,121) by P1 transduction (Miller, J. H. Experimentsin Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview,N.Y.) to obtain the strain 57-Δtarget gene. The strain 57 has beendeposited in the Russian National Collection of IndustrialMicroorganisms (VKPM) (Russia, 117545 Moscow, 1^(st) Dorozhny proezd, 1)on May 19, 1997 under accession number VKPM B-7386.

Both E. coli strains, 57 and 57-Δtarget gene, can be cultured for 18-24hours at 37° C. on L-agar plates. To obtain a seed culture, the strainscan be grown on a rotary shaker (250 rpm) at 32° C. for 18 hours in20×200-mm test tubes containing 2 ml of L-broth supplemented with 4%sucrose. Then, the fermentation medium can be inoculated with 0.21 ml ofseed material (10%). The fermentation can be performed in 2 ml of aminimal fermentation medium in 20×200-mm test tubes. Cells can be grownfor 48-72 hours at 32° C. with shaking at 250 rpm. The amount ofL-leucine can be measured by paper chromatography (liquid phasecomposition: butanol-acetic Acid-water=4:1:1).

The composition of the fermentation medium (g/l) (pH 7.2) is as follows:

Glucose 60.0 (NH₄)₂SO₄ 25.0 K₂HPO₄ 2.0 MgSO₄7H₂O 1.0 Thiamine 0.01 CaCO₃25.0

Glucose and CaCO₃ are sterilized separately.

Example 6 Production of L-Histidine by E. coli Strain Having a GeneCoding for sRNA Deleted

To test the effect of inactivation of a gene coding for sRNA onL-histidine production, DNA fragments from the chromosome of theabove-described mutant E. coli MG1655 having a gene coding for sRNA(target gene) deleted and marked with the Cm resistance gene can betransferred to the histidine-producing E. coli strain 80 by P1transduction (Miller, J. H. Experiments in Molecular Genetics, ColdSpring Harbor Lab. Press, 1972, Plainview, N.Y.) to obtain strain80-Δtarget gene. The strain 80 has been described in Russian patent2119536 and deposited in the Russian National Collection of IndustrialMicroorganisms (Russia, 117545 Moscow, 1st Dorozhny proezd, 1) on Oct.15, 1999 under accession number VKPM B-7270 and then converted to adeposit under the Budapest Treaty on Jul. 12, 2004.

Both E. coli strains, 80 and 80-Δtarget gene, can each be cultured inL-broth for 6 h at 29° C. Then, 0.1 ml of obtained culture can beinoculated into 2 ml of fermentation medium in a 20×200-mm test tube andcultivated for 65 hours at 29° C. with shaking on a rotary shaker (350rpm). After cultivation, the amount of histidine which accumulates inthe medium can be determined by paper chromatography. The paper can bedeveloped with a mobile phase consisting of n-butanol:aceticacid:water=4:1:1 (v/v). A solution of ninhydrin (0.5%) in acetone can beused as a visualizing reagent.

The composition of the fermentation medium (g/l) is as follows (pH 6.0):

Glucose 100.0 Mameno (soybean hydrolysate) 0.2 of as total nitrogenL-proline 1.0 (NH₄)₂SO₄ 25.0 KH₂PO₄ 2.0 MgSO₄7H₂0 1.0 FeSO₄7H₂0 0.01MnSO₄ 0.01 Thiamine 0.001 Betaine 2.0 CaCO₃ 60.0

Glucose, proline, betaine and CaCO₃ are sterilized separately. The pH isadjusted to 6.0 before sterilization.

Example 7 Production of L-Glutamate by E. coli Strain Having a GeneCoding for sRNA Deleted

To test the effect of inactivation of a gene coding for sRNA onL-glutamate production, DNA fragments from the chromosome of theabove-described mutant E. coli MG1655 having a gene coding for sRNA(target gene) deleted and marked with the Cm resistance gene can betransferred to the E. coli L-glutamate-producing strain VL334thrC⁺ (EP1172433) by P1 transduction (Miller, J. H. Experiments in MolecularGenetics, Cold Spring Harbor Lab. Press, 1972, Plainview, N.Y.) toobtain the strain VL334thrC⁺-Δtarget gene. The strain VL334thrC⁺ hasbeen deposited in the Russian National Collection of IndustrialMicroorganisms (VKPM) (Russia, 117545 Moscow, 1^(st) Dorozhny proezd, 1)on Dec. 6, 2004 under the accession number VKPM B-8961 and thenconverted to a deposit under the Budapest Treaty on Dec. 8, 2004.

Both strains, VL334thrC⁺ and VL334thrC-Δtarget gene, can be grown for18-24 hours at 37° C. on L-agar plates. Then, one loop of the cells canbe transferred into test tubes containing 2 ml of fermentation medium.The fermentation medium contains glucose (60 g/l), ammonium sulfate (25g/l), KH₂PO₄ (2 g/l), MgSO₄ (1 g/l), thiamine (0.1 mg/ml), L-isoleucine(70 μg/ml), and CaCO₃ (25 g/l). The pH is adjusted to 7.2. Glucose andCaCO₃ are sterilized separately. Cultivation can be carried out at 30°C. for 3 days with shaking. After the cultivation, the amount ofL-glutamic acid which is produced can be determined by paperchromatography (liquid phase composition of butanol-aceticacid-water=4:1:1) with subsequent staining by ninhydrin (1% solution inacetone) and further elution of the compounds in 50% ethanol with 0.5%CdCl₂.

Example 8 Production of L-Phenylalanine by E. coli Strain Having a GeneCoding for sRNA Deleted

To test the effect of inactivation of a gene coding for sRNA onL-phenylalanine production, DNA fragments from the chromosome of theabove-described mutant E. coli MG1655 having a gene coding for sRNA(target gene) deleted and marked with the Cm resistance gene can betransferred to the phenylalanine-producing E. coli strain AJ12739 by P1transduction (Miller, J. H. Experiments in Molecular Genetics, ColdSpring Harbor Lab. Press, 1972, Plainview, N.Y.) to obtain strainAJ12739-Δtarget gene. The strain AJ12739 has been deposited in theRussian National Collection of Industrial Microorganisms (VKPM) (Russia,117545 Moscow, 1^(st) Dorozhny proezd, 1) on Nov. 6, 2001 underaccession no. VKPM B-8197 and then converted to a deposit under theBudapest Treaty on Aug. 23, 2002.

Both strains, AJ12739-Δtarget gene and AJ12739, can be cultivated at 37°C. for 18 hours in a nutrient broth, and 0.3 ml of the obtained culturecan each be inoculated into 3 ml of a fermentation medium in a 20×200-mmtest tube and cultivated at 37° C. for 48 hours with shaking on a rotaryshaker. After cultivation, the amount of phenylalanine which accumulatesin the medium can be determined by TLC. The 10×15-cm TLC plates coatedwith 0.11-mm layers of Sorbfil silica gel containing no fluorescentindicator (Stock Company Sorbpolymer, Krasnodar, Russia) can be used.The Sorbfil plates can be developed with a mobile phase consisting ofpropan-2-ol:ethylacetate:25% aqueous ammonia:water=40:40:7:16 (v/v). Asolution of ninhydrin (2%) in acetone can be used as a visualizingreagent.

The composition of the fermentation medium (g/l) is as follows:

Glucose 40.0 (NH₄)₂SO₄ 16.0 K₂HPO₄ 0.1 MgSO₄7H₂O 1.0 FeSO₄7H₂O 0.01MnSO₄5H₂O 0.01 Thiamine HCl 0.0002 Yeast extract 2.0 Tyrosine 0.125CaCO₃ 20.0

Glucose and magnesium sulfate are sterilized separately. CaCO₃ isdry-heat sterilized at 180° for 2 hours. The pH is adjusted to 7.0.

Example 9 Production of L-Tryptophan by E. coli Strain Having a GeneCoding for sRNA Deleted

To test the effect of inactivation of a gene coding for sRNA onL-tryptophan production, DNA fragments from the chromosome of theabove-described mutant E. coli MG1655 having a gene coding for sRNA(target gene) deleted and marked with the Cm resistance gene can betransferred to the tryptophan-producing E. coli strain SV164 (pGH5) byP1 transduction (Miller, J. H. Experiments in Molecular Genetics, ColdSpring Harbor Lab. Press, 1972, Plainview, N.Y.) to obtain the strainSV164 (pGH5)-Δtarget gene. The strain SV164 has the trpE allele encodinganthranilate synthase free from feedback inhibition by tryptophan. Theplasmid pGH5 harbors a mutant serA gene encoding phosphoglyceratedehydrogenase free from feedback inhibition by serine. The strain SV164(pGH5) was described in detail in U.S. Pat. No. 6,180,373 or Europeanpatent 0662143. The strains SV164 (pGH5)-ΔgcvB::cat, SV164 (pGH5)ΔrprA::cat, SV164 (pGH5)-ΔsraA::cat, SV164 (pGH5)-ΔsraB::cat, SV164(pGH5)-ΔsraH::cat and SV164 (pGH5)-ΔsroE::cat were obtained in thismanner.

Strains SV164 (pGH5) and each of strains SV164 (pGH5)-ΔgcvB::cat, SV164(pGH5)-ΔrprA::cat, SV164 (pGH5)-ΔsraA::cat, SV164 (pGH5)-ΔsraB::cat,SV164 (pGH5)-ΔsraH::cat or SV164 (pGH5)-ΔsroE::cat, were separatelycultivated with shaking at 32° C. for 18 hours in 3 ml of nutrient brothsupplemented with tetracycline (10 mg/ml, marker of pGH5 plasmid). Theobtained cultures (0.3 ml each) were inoculated into 3 ml of afermentation medium containing tetracycline (10 mg/ml) in 20×200-mm testtubes, and cultivated at 32° C. for 72 hours with a rotary shaker at 250rpm. After cultivation, the amount of tryptophan which accumulates inthe medium was determined by TLC as described in Example 8. The resultsof ten (for SV164 (pGH5)-ΔgcvB::cat, SV164 (pGH5)-ΔrprA::cat and SV164(pGH5)-ΔsraB::cat)/seven (for SV164 (pGH5)-ΔsraA::cat and SV164(pGH5)-ΔsraH::cat)/five (for SV164 (pGH5)-ΔsroE::cat) independent testtube fermentations are shown in Table 4. As follows from Table 4, SV164(pGH5)-ΔgcvB::cat, SV164 (pGH5)-ΔrprA::cat, SV164 (pGH5)-ΔsraA::cat,SV164 (pGH5)-ΔsraB::cat, SV164 (pGH5)-ΔsraH::cat and SV164(pGH5)-ΔsroE::cat produced a higher amount of L-tryptophan, as comparedto SV164 (pGH5).

TABLE 4 Amount of L- Strain OD₅₄₀ tryptophane, g/l SV164/pGH5 34.6 ± 1.64.1 ± 0.1 SV164/pGH5-ΔrprA 34.8 ± 1.4 4.4 ± 0.4 SV164/pGH5-ΔsraB 32.9 ±0.9 4.6 ± 0.5 SV164 (pGH5) 38.5 ± 0.3 3.9 ± 0.4 SV164 (pGH5)-ΔsraH 34.1± 1.1 4.3 ± 0.9 SV164 (pGH5) 34.2 ± 1.8 3.3 ± 0.3 SV164 (pGH5)-ΔsraA23.3 ± 1.2 3.8 ± 0.6 SV164 (pGH5) 28.6 ± 0.5 6.3 ± 0.2 SV164(pGH5)-ΔsroE 26.3 ± 1.1 6.6 ± 0.1 SV164 (pGH5)-ΔgcvB 28.7 ± 0.5 6.6 ±0.2

The fermentation medium components are listed in Table 3, but should besterilized in separate groups (A, B, C, D, E, F, and G), as shown, toavoid adverse interactions during sterilization.

TABLE 3 Solutions Component Final concentration, g/l A KH₂PO₄ 0.28 NaCl0.14 (NH₄)₂SO₄ 16 L-Methionine 0.08 L-Phenylalanine 0.28 L-Tyrosine 0.28Mameno (total N) 0.07 B Glucose 40.0 MgSO₄7H₂O 0.03 C FeSO₄7H₂O 0.03 DNa₂MoO₄2H₂O 0.00015 H₃BO₃ 0.0025 CoCl₂6H₂O 0.00007 CuSO₄5H₂O 0.00025MnCl₂4H₂O 0.0016 ZnSO₄7 H₂O 0.0003 E Thiamine HCl 0.001 F CaCO₃ 30.0 GPyridoxine 0.03 The pH of solution A is adjusted to 7.1 with NH₄OH.

Example 10 Production of L-Proline by E. coli Strain Having a GeneCoding for sRNA Deleted

To test the effect of inactivation of a gene coding for sRNA onL-proline production, DNA fragments from the chromosome of theabove-described mutant E. coli MG1655 having a gene coding for sRNA(target gene) deleted and marked with the Cm resistance gene can betransferred to the proline-producing E. coli strain 702ilvA by P1transduction (Miller, J. H. Experiments in Molecular Genetics, ColdSpring Harbor Lab. Press, 1972, Plainview, N.Y.) to obtain strain702ilvA-Δtarget gene. The strain 702ilvA has been deposited in theRussian National Collection of Industrial Microorganisms (VKPM) (Russia,117545 Moscow, 1^(st) Dorozhny proezd, 1) on Jul. 18, 2000 underaccession number VKPM B-8012 and then converted to a deposit under theBudapest Treaty on May 18, 2001.

Both E. coli strains, 702ilvA and 702ilvA-Δtarget gene, can be grown for18-24 hours at 37° C. on L-agar plates. Then, these strains can becultivated under the same conditions as in Example 7.

Example 11 Production of L-Arginine by E. coli Strain Having a GeneCoding for sRNA Deleted

To test the effect of inactivation of a gene coding for sRNA onL-arginine production, DNA fragments from the chromosome of theabove-described mutant E. coli MG1655 having a gene coding for sRNA(target gene) deleted and marked with the Cm resistance gene can betransferred to the arginine-producing E. coli strain 382 by P1transduction (Miller, J. H. Experiments in Molecular Genetics, ColdSpring Harbor Lab. Press, 1972, Plainview, N.Y.) to obtain strain382-Δtarget gene. The strain 382 has been deposited in the RussianNational Collection of Industrial Microorganisms (VKPM) (Russia, 117545Moscow, 1^(st) Dorozhny proezd, 1) on Apr. 10, 2000 under accessionnumber VKPM B-7926 and then converted to a deposit under the BudapestTreaty on May 18, 2001. The strains 382-ΔgcvB::cat, 382-ΔrprA::cat,382-ΔrygB-sraE::cat and 382-ΔsraH::cat were obtained in this manner.

Strains 382 and each of strains 382-ΔgcvB::cat, 382-ΔrprA::cat,382-ΔrygB-sraE::cat or 382-ΔsraH::cat were separately cultivated withshaking at 37° C. for 18 hours in 3 ml of nutrient broth, and 0.3 ml ofthe obtained cultures were inoculated into 2 ml of a fermentation mediumin 20×200-mm test tubes and cultivated at 32° C. for 48 hours on arotary shaker.

After the cultivation, the amount of L-arginine which had accumulated inthe medium was determined by paper chromatography using the followingmobile phase: butanol:acetic acid:water=4:1:1 (v/v). A solution ofninhydrin (2%) in acetone was used as a visualizing reagent. A spotcontaining L-arginine was cut out, L-arginine was eluted with 0.5% watersolution of CdCl₂, and the amount of L-arginine was estimatedspectrophotometrically at 540 nm. The results of ten (for 382-ΔgcvB::catand 382-ΔrprA::cat)/nine (for 382-ΔrygB-sraE::cat and 382-ΔsraH::cat)independent test tube fermentations are shown in Table 5. As followsfrom Table 5, 382-ΔgcvB::cat, 382-ΔrprA::cat, 382-ΔrygB-sraE::cat and382-ΔsraH::cat produced a higher amount of L-arginine, as compared to382.

TABLE 5 Amount of Strain OD₅₄₀ L-arginine, g/l 382 13.0 ± 2.2  9.7 ± 1.3382-ΔgcvB 12.9 ± 1.6 10.9 ± 0.8 382 13.8 ± 0.7  8.6 ± 0.4 382-ΔrprA 13.4± 0.7 10.1 ± 0.8 382 14.2 ± 0.9 11.1 ± 0.7 382-ΔsraH 15.0 ± 0.9 11.9 ±0.7 382-ΔrygB-sraE 14.0 ± 0.7 12.6 ± 0.4

The composition of the fermentation medium (g/l) was as follows:

Glucose 48.0 (NH4)₂SO₄ 35.0 KH₂PO₄ 2.0 MgSO₄7H₂O 1.0 Thiamine HCl 0.0002Yeast extract 1.0 L-isoleucine 0.1 CaCO₃ 5.0

Glucose and magnesium sulfate were sterilized separately. CaCO₃ wasdry-heat sterilized at 180° C. for 2 hours. The pH was adjusted to 7.0.

Example 12 Elimination of Cm Resistance Gene (Cat Gene) from theChromosome of L-Amino Acid-Producing E. coli Strains

The Cm resistance gene (cat gene) can be eliminated from the chromosomeof the L-amino acid-producing strain using the int-xis system. For thatpurpose, an L-amino acid-producing strain having DNA fragments from thechromosome of the above-described E. coli strain MG1655 Δtargetgene::cat transferred by P1 transduction can be transformed with plasmidpMWts-Int/Xis. Transformant clones can be selected on the LB-mediumcontaining 100 μg/ml of ampicillin. Plates can be incubated overnight at30° C. Transformant clones can be cured from the cat gene by spreadingthe separate colonies at 37° C. (at that temperature repressor CIts ispartially inactivated and transcription of the int/xis genes isderepressed) followed by selection of Cm^(S)Ap^(R) variants. Eliminationof the cat gene from the chromosome of the strain can be verified byPCR. Locus-specific primers P3 and P4 (see Table 1) can be used in PCRfor the verification. Conditions for PCR verification can be asdescribed above. The PCR product obtained in reaction with cells havingthe eliminated cat gene as a template should be much shorter (˜0.3-0.5kbp in length) then strain with cat gene.

Example 13 Production of L-Lysine Using an Escherichia Bacterium inwhich Expression of rygB-sraE Gene is Attenuated

In order to evaluate the effect of rygB-sraE gene attenuation onL-lysine yield, a rygB-sraE-disrupted strain was constructed. The wholegenome sequence of Escherichia coli K-12 strain has been disclosed(Science, 277, 1453-1474 (1997)). The nucleotide sequence of rygB-sraEgene is also disclosed, and the strain can be constructed based on thesequence.

(1) Disruption of the rygB-sraE Gene

The rygB-sraE gene was disrupted according to the method called“Red-driven integration” that was developed by Datsenko and Wanner(Proc. Natl. Acad. Sci. USA, 2000, vol. 97, No. 12, p 6640-6645).“Red-driven integration” makes it possible to construct a gene-disruptedstrain in one step by employing a PCR product obtained by using asprimers synthetic oligonucleotides designed to have a part of thetargeted gene on the 5′-ends and a part of an antibiotic-resistance geneon the 3′-ends. According to this method, primers which arecomplementary to a region proximal to the rygB-sraE gene and to a regionproximal to a gene conferring antibiotic resistance to a templateplasmid, respectively, were designed. PCR was performed using theoligonucleotides of SEQ ID NOS: 117 and 118 and the plasmidpMW118-att-cat as a template.

The obtained PCR product was purified by using an agarose gel, and usedto transform the E. coli WC196LC strain that carries a plasmid pKD46that has temperature-sensitive replication ability. pKD46 contains a DNAfragment of 2,154 nucleotides derived from λ phage which contains theRed recombinase-encoding genes (γ, β, and exo genes) of the λ Redhomologous recombination system, which is controlled by anarabinose-inducible ParaB promoter (GenBank/EMBL Accession No. J02459,nucleotide numbers 31088 to 33241). pKD46 is necessary to integrate thePCR product into the chromosome of the WC196LC strain. The WC196LCstrain (WO2006/038695) is an L-lysine-producing strain obtained bydisrupting lysine decarboxylase genes (cadA and ldcC) in the WC196strain (FERM BP-5252).

Competent cells for electroporation were prepared as follows. E. coliWC196LC/pKD46 strain was cultured overnight at 30° C. in LB medium thatcontained 100 mg/L of ampicillin, and then was diluted to 100-fold with5 ml of SOB medium (Molecular Cloning Laboratory Manual 2nd Edition,Sambrook, J. et al., Cold Spring Harbor Laboratory Press (1989)) thatcontained ampicillin and L-arabinose (1 mM). The diluted cells weregrown at 30° C. with aeration until OD600 became approximately 0.6, andthen concentrated to 100-fold and washed with ice-cold 10% glycerolsolution three times, which were then used for electroporation.Electroporation was performed using 40 μL of the competent cells andabout 100 ng of the PCR product. After electroporation, the cells wereadded to 1 mL of SOC medium (Molecular Cloning Laboratory Manual 2ndEdition, Sambrook, J. et al., Cold Spring Harbor Laboratory Press(1989)) and cultured at 37° C. for 1 hour, and then cultured overnightat 37° C. on L-agar medium containing chloramphenicol to select achloramphenicol-resistant recombinant strain. Then, in order to cure thepKD46 plasmid, the strain was subjected to passage culture twice at 42°C. on L-agar medium containing chloramphenicol, and theampicillin-resistance of the obtained strains was tested to select anampicillin-sensitive strain due to curing of the pKD46 plasmid.

The disruption of the rygB-sraE gene in the obtained strain wasconfirmed by PCR based on the presence of the chloramphenicol-resistantgene. The obtained rygB-sraE-disrupted strain was namedWC196LCΔrygB-sraE.

(2) The Effect of rygB-sra Gene Disruption on the E. coliL-Lysine-Producing Strain

According to a conventional method, the WC196LC strain andWC196LCΔrygB-sraE strain were transformed with the plasmid pCABD2(WO01/53459), which is a plasmid for L-lysine production containing thedapA gene, dapB gene, lysC gene and ddh gene, to prepare aWC196LC/pCABD2 strain and WC196LCΔrygB-sraE/pCABD2 strain.

The WC196LC/pCABD2 strain and WC196LCΔrygB-sraE/pCABD2 strain werecultured at 37° C. in L medium contains 20 mg/L of streptomycin untilOD600 became approximately 0.6, and then added to an equal volume of 40%glycerol solution and mixed. Then, the mixture was dispensed in anappropriate volume and stocked at −80° C., which was used as a glycerolstock.

The glycerol stocks of these strains were thawed, and 100 μl of each ofthe strains was spread uniformly over an L plate containing 20 mg/L ofstreptomycin and cultured at 37° C. for 24 hours. About one eighth ofthe cells of each strain on the plate was inoculated into 20 mL of thefermentation medium that has the composition shown below and furthercontains 20 mg/L of streptomycin in 500 mL-volume Sakaguchi flask, andcultured at 37° C. for 24 hours with reciprocal shaker. The amount ofL-lysine that accumulated in the medium was analyzed by using BiotechAnalyze AS210 (Sakura Seiki).

Table 6 shows OD600 and L-lysine accumulation after 24 hour culture. Itwas revealed from this result that L-lysine accumulation was improved bydisrupting the rygB-sraE gene.

L-Lysine Production Medium:

Glucose  40 g/L (NH₄)₂SO₄  24 g/L KH₂PO₄ 1.0 g/L MgSO₄•7H₂O 1.0 g/LFeSO₄•7H₂O 0.01 g/L  MnSO₄•5H₂O 0.01 g/L  Yeast extract 2.0 g/L Calciumcarbonate (Official grade)  30 g/L

The medium was adjusted to pH 7.0 with potassium hydroxide andsterilized by steam at 115° C. for 10 minutes.

Glucose and MgSO₄.7H₂O were separately sterilized.

Calcium carbonate (Official grade) was separately sterilized by heatingat 180° C.

TABLE 6 L-lysine accumulation Strain OD600 (g/L) WC196LC/pCABD2 14.110.5 WC196LCΔrygB- 18.1 12.8 sraE/pCABD2 *The amount of L-lysineaccumulation was shown by the amount of hydrochloride salt of L-lysine.

Example 14 Production of L-Lysine Using an Escherichia Bacterium inwhich Expression of gcvB Gene is Attenuated

In order to evaluate the effect of gcvB gene attenuation on L-lysineyield, a gcvB-disrupted strain was constructed. The whole genomesequence of Escherichia coli K-12 strain has been disclosed (Science,277, 1453-1474 (1997)). The nucleotide sequence of gcv gene is alsodisclosed, and the strain can be constructed based on the sequence.

(1) Disruption of the gcvB Gene

The gcvB gene was disrupted according to the method called “Red-drivenintegration” that was developed by Datsenko and Wanner (Proc. Natl.Acad. Sci. USA, 2000, vol. 97, No. 12, p 6640-6645). According to thismethod, primers which are complementary to a region proximal to the gcvBgene and to a region proximal to a gene conferring antibiotic resistanceto a template plasmid, respectively, were designed. PCR was performedusing the oligonucleotides of SEQ ID NOS: 119 and 120 and the plasmidpMW118-att-cat as a template.

The obtained PCR product was purified by using an agarose gel, and usedto transform the E. coli WC196LC strain that carries a plasmid pKD46that has temperature-sensitive replication ability.

Competent cells for electroporation were prepared as follows. E. coliWC196LC/pKD46 strain was cultured overnight at 30° C. in LB medium thatcontained 100 mg/L of ampicillin, and then was diluted to 100-fold with5 ml of SOB medium that contained ampicillin and L-arabinose (1 mM). Thediluted cells were grown at 30° C. with aeration until OD600 becameapproximately 0.6, and then concentrated to 100-fold and washed withice-cold 10% glycerol solution three times, which were used forelectroporation. Electroporation was performed using 40 μL of thecompetent cells and about 100 ng of the PCR product. Afterelectroporation, the cells were added to 1 mL of SOC medium and culturedat 37° C. for 1 hour, and then cultured overnight at 37° C. on L-agarmedium containing chloramphenicol to select a chloramphenicol-resistantrecombinant strain. Then, in order to cure the pKD46 plasmid, the strainwas subjected to passage culture twice at 42° C. on L-agar mediumcontaining chloramphenicol, and the ampicillin-resistance of theobtained strains was tested to select an ampicillin-sensitive strain dueto curing of the pKD46 plasmid.

The disruption of the gcvB gene in the obtained strain was confirmed byPCR based on the presence of the chloramphenicol-resistant gene. Theobtained gcvB-disrupted strain was named WC196LCΔgcvB.

(2) The Effect of gcvB Gene Disruption on an E. coli L-Lysine-ProducingStrain

According to a conventional method, the WC196LC strain and WC196LCΔgcvBstrain were transformed with the plasmid pCABD2 to prepare aWC196LC/pCABD2 strain and WC196LCΔgcvB/pCABD2 strain.

The WC196LC/pCABD2 strain and WC196LCΔgcvB/pCABD2 strain were culturedat 37° C. in L medium containing 20 mg/L of streptomycin until OD600became approximately 0.6, and then added to an equal volume of 40%glycerol solution and mixed. Then, the mixture was dispensed in anappropriate volume and stocked at −80° C., which was used as a glycerolstock.

The glycerol stocks of these strains were thawed, and 100 μl of each ofthe strains was spread uniformly over an L plate containing 20 mg/L ofstreptomycin and cultured at 37° C. for 24 hours. About one eighth ofthe cells of each strain on the plate was inoculated into 20 mL of thefermentation medium that has a composition shown below and furthercontains 20 mg/L of streptomycin in 500 mL-volume Sakaguchi flask, andcultured at 37° C. for 24 hours with reciprocal shaker. The amount ofL-lysine that accumulated in the medium was analyzed by using BiotechAnalyze AS210 (Sakura Seiki).

Table 7 shows OD600 and L-lysine accumulation after 24 hour culture. Itwas revealed from this result that L-lysine accumulation was improved bydisrupting the gcvB gene.

TABLE 7 L-lysine accumulation Strain OD600 (g/L) WC196LC/pCABD2 14.110.5 WC196LCΔgcvB/pCABD2 18.6 13.4 *The amount of L-lysine accumulationwas shown by the amount of hydrochloride salt of L-lysine.

While the invention has been described in detail with reference topreferred embodiments thereof, it will be apparent to one skilled in theart that various changes can be made, and equivalents employed, withoutdeparting from the scope of the invention. All the cited referencesherein are incorporated as a part of this application by reference.

1. A method for producing an L-amino acid comprising: cultivating an Escherichia coli in a medium, and collecting said L-amino acid from the medium, wherein said Escherichia coli has been modified to attenuate expression of sraD gene, wherein said L-amino acid is selected from the group consisting of L-threonine, L-lysine, and combinations thereof.
 2. The method according to claim 1, wherein said sraD gene comprises a nucleotide sequence that hybridizes with a complement of the nucleotide sequence of SEQ ID NO: 98 under stringent conditions comprising washing at 0.1×SSC, 0.1% SDS at 60° C.
 3. The method according to claim 1, wherein said sraD gene comprises the nucleotide sequence of SEQ ID NO:
 98. 